Therapeutic cd47 antibodies

ABSTRACT

Provided are anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and to methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, inflammatory diseases or as diagnostics for determining the level of CD47 in tissue samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2016/052383, filed Sep. 17, 2016, which claims the benefit ofUnited States Provisional Application Nos. 62/220,691, filed Sep. 18,2015; 62/263,544, filed Dec. 4, 2015; 62/221,852, filed Sep. 22, 2015;62/220,725, filed Sep. 18, 2015; 62/232,681, filed Sep. 25, 2015;62/252,171, filed Nov. 6, 2015; and 62/354,592, filed Jun. 24, 2016; thedisclosures of which are hereby incorporated by reference as if writtenherein in their entireties.

FIELD OF THE DISCLOSURE

This disclosure is related generally to anti-CD47 monoclonal antibodies(anti-CD47 mAbs) with distinct functional profiles as described herein,methods to generate anti-CD47 mAbs, and to methods of using theseanti-CD47 mAbs as therapeutics for the prevention and treatment of solidand hematological cancers, ischemia-reperfusion injury, cardiovasculardiseases, autoimmune diseases, or inflammatory diseases or asdiagnostics for determining the level of CD47 in tissue samples.

BACKGROUND OF THE DISCLOSURE

CD47 is a cell surface receptor comprised of an extracellular IgV setdomain, a 5 transmembrane domain, and a cytoplasmic tail that isalternatively spliced. Two ligands bind CD47: signal inhibitory receptorprotein α (SIRPα) and thrombospondin-1 (TSP1). CD47 expression and/oractivity have been implicated in a number of diseases and disorders.Accordingly, there exists a need for therapeutic compositions andmethods for treating diseases and conditions associated with CD47 inhumans and animals, including the prevention and treatment of solid andhematological cancers, ischemia-reperfusion injury (IRI), cardiovasculardiseases, or an autoimmune or inflammatory disease. There also exists aneed for diagnostic compositions and methods for determining the levelof CD47 expression in tumor samples.

SUMMARY OF THE DISCLOSURE

The present disclosure describes anti-CD47 mAbs with distinct functionalprofiles. These antibodies possess distinct combinations of propertiesselected from the following: 1) exhibit cross-reactivity with one ormore species homologs of CD47; 2) block the interaction between CD47 andits ligand SIRPα; 3) increase phagocytosis of human tumor cells, 4)induce death of susceptible human tumor cells; 5) do not induce celldeath of human tumor cells; 6) have reduced binding to human red bloodcells (hRBCs); 7) have no detectable binding to hRBCs; 8) cause reducedagglutination of hRBCs; 9) cause no detectable agglutination of hRBCs;10) reverse TSP1 inhibition of the nitric oxide (NO) pathway and/or 11)do not reverse TSP1 inhibition of the NO pathway. The antibodies of thedisclosure are useful in various therapeutic methods for treatingdiseases and conditions associated with CD47 in humans and animals,including the prevention and treatment of solid and hematologicalcancers, autoimmune diseases, inflammatory diseases, IRI, andcardiovascular diseases. The antibodies of the disclosure are alsouseful as diagnostics to determine the level of CD47 expression intissue samples. Embodiments of the disclosure include isolatedantibodies and immunologically active binding fragments thereof;pharmaceutical compositions comprising one or more of the anti-CD47monoclonal antibodies, preferably chimeric or humanized forms of saidantibodies; methods of therapeutic use of such anti-CD47 monoclonalantibodies; and cell lines that produce these anti-CD47 monoclonalantibodies.

The embodiments of the disclosure include the mAbs, or antigen-bindingfragments thereof, which are defined by reference to specific structuralcharacteristics i.e. specified amino acid sequences of either the CDRsor entire heavy chain or light chain variable domains. All of theseantibodies bind to CD47.

The monoclonal antibodies, or antigen binding fragments thereof maycomprise at least one, usually at least three, CDR sequences as providedherein, usually in combination with framework sequences from a humanvariable region or as an isolated CDR peptide. In some embodiments, anantibody comprises at least one light chain comprising the three lightchain CDR sequences provided herein situated in a variable regionframework, which may be, without limitation, a murine or human variableregion framework, and at least one heavy chain comprising the threeheavy chain CDR sequences provided herein situated in a variable regionframework, which may be, without limitation, a human or murine variableregion framework.

Preferred embodiments are anti-CD47 mAbs, or antigen binding fragmentsthereof, comprising a heavy chain variable domain comprising a variableheavy chain CDR1, variable heavy chain CDR2, and a variable heavy chainCDR3, wherein said variable heavy chain CDR1 comprises an amino acidsequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3; said variable heavy chain CDR2 comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6; and said variable heavy chain CDR3 comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10.

The heavy chain variable domain may comprise any one of the listedvariable heavy chain CDR1 sequences (HCDR1) in combination with any oneof the variable heavy chain CDR2 sequences (HCDR2) and any one of thevariable heavy chain CDR3 sequences (HCDR3). However, certainembodiments of HCDR1 and HCDR2 and HCDR3 are particularly preferred,which derive from a single common V_(H) domain, examples of which aredescribed herein.

The antibody or antigen binding fragment thereof may additionallycomprise a light chain variable domain (V_(L)), which is paired with theV_(H) domain to form an antigen binding domain. Preferred light chainvariable domains are those comprising a variable light chain CDR1,variable light chain CDR2, and a variable light chain CDR3, wherein saidvariable light chain CDR1 comprises an amino acid sequence selected fromthe group consisting of:

SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14; said variablelight chain CDR2 optionally comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17;and said variable light chain CDR3 optionally comprises an amino acidsequence selected from the group consisting of: SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20.

The light chain variable domain may comprise any one of the listedvariable light chain CDR1 sequences (LCDR1) in combination with any oneof the variable light chain CDR2 sequences (LCDR2) and any one of thevariable light chain CDR3 sequences (LCDR3). However, certainembodiments of LCDR1 and LCDR2 and LCDR3 are particularly preferred,which derive from a single common V_(L) domain, examples of which aredescribed herein.

Any given CD47 antibody or antigen binding fragment thereof comprising aVH domain paired with a VL domain will comprise a combination of 6 CDRs:variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2 (HCDR2),variable heavy chain CDR3 (HCDR3), variable light chain CDR1 (LCDR1),variable light chain CDR2 (LCDR2), and variable light chain CDR1(LCDR1). Although all combinations of 6 CDRs selected from the CDRsequence groups listed above are permissible, and within the scope ofthe disclosure, certain combinations of 6 CDRs are particularlypreferred.

Preferred combinations of 6 CDRs include, but are not limited to, thecombinations of variable heavy chain CDR1 (HCDR1), variable heavy chainCDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chainCDR1 (LCDR1), variable light chain CDR2 (LCDR2), and variable lightchain CDR3 (LCDR3) selected from the group consisting of:

-   -   (i) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:4,        HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO:11,        LCDR2 comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:18;    -   (ii) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:4,        HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO:11,        LCDR2 comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:18;    -   (iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID        NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID        NO:12, LCDR2 comprising SEQ ID NO:16, LCDR3 comprising SEQ ID        NO:19;    -   (iv) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5,        HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:13,        LCDR2 comprising SEQ ID NO:16, LCDR3 comprising SEQ ID NO:19;        and    -   (v) HCDR1 comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6,        HCDR3 comprising SEQ ID NO:10, LCDR1 comprising SEQ ID NO:14,        LCDR2 comprising SEQ ID NO:17, LCDR3 comprising SEQ ID NO:20.

Further preferred anti-CD47 antibodies include antibodies or antigenbinding fragments thereof, comprising a heavy chain variable domainhaving an amino acid sequence selected from the group consisting of: theamino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQID NO:40 and amino acid sequences exhibiting at least 90%, 95%, 97%,98%, or 99% sequence identity to one of the recited sequences.Alternatively or in addition, preferred anti-CD47 antibodies includingantibodies or antigen binding fragments thereof may comprise a lightchain variable domain having an amino acid sequence selected from thegroup consisting of: the amino acid sequences of SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52 and amino acidsequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequenceidentity to one of the recited sequences.

Although all possible pairing of V_(H) domains and V_(L) domainsselected from the V_(H) and VL domain sequence groups listed above arepermissible, and within the scope of the disclosure, certaincombinations of V_(H) and V_(L) domains are particularly preferred.Accordingly, preferred CD47 antibodies, or antigen binding fragmentsthereof, are those comprising a combination of a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)), wherein thecombination is selected from the group consisting of:

-   -   (i) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:21 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:41;    -   (ii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:23 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (iii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:34 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:49;    -   (iv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:36 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (v) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:38 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (vi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:39 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (vii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:24 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (viii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:37 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (ix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:33 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (x) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:26 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:44;    -   (xi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:27 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:44; and    -   (xii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:38 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:39 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:40 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (xv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:36 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xvi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xvii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xviii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xx) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:33 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xxi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:26 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (xxvi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:27 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (xxvii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:28 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:46;    -   (xxviii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:35 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:50;    -   (xxix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxx) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:37 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51; and    -   (xxxiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:40 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51.

Preferred anti-CD47 antibodies or antigen binding fragments thereof mayalso comprise a combination of a heavy chain variable domain and a lightchain variable domain wherein the heavy chain variable domain comprisesa VH sequence with at least 85% sequence identity, or at least 90%sequence identity, or at least 95% sequence identity, or at least 97%,98% or 99% sequence identity, to the heavy chain amino acid sequencesshown above in (i) to (xxxiv) and/or the light chain variable domaincomprises a VL sequence with at least 85% sequence identity, or at least90% sequence identity, or at least 95% sequence identity, or at least97%, 98% or 99% sequence identity, to the light chain amino acidsequences shown above in (i) to (xxxiv). The specific VH and VL pairingsor combinations in parts (i) through (xxxiv) may be preserved foranti-CD47 antibodies having VH and VL domain sequences with a particularpercentage sequence identity to these reference sequences.

For all embodiments wherein the heavy chain and/or light chain variabledomains of the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, the VHand/or VL domains may retain identical CDR sequences to those present inthe reference sequence such that the variation is present only withinthe framework regions.

In another embodiment, the preferred CD47 antibodies, or antigen bindingfragments thereof, are those comprising a combination of a heavy chain(HC) and a light chain (LC), wherein the combination is selected fromthe group consisting of:

-   -   (i) a heavy chain comprising the amino acid sequence of SEQ ID        NO:78 and a light chain comprising the amino acid sequence SEQ        ID NO:67;    -   (ii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:79 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (iii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:80 and a light chain comprising the amino acid sequence SEQ        ID NO:70;    -   (iv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:81 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (v) a heavy chain comprising the amino acid sequence of SEQ ID        NO:82 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (vi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:83 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (vii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:84 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (viii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:85 and a light chain comprising the amino acid sequence        SEQ ID NO:71;    -   (ix) a heavy chain comprising the amino acid sequence of SEQ ID        NO:86 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (x) a heavy chain comprising the amino acid sequence of SEQ ID        NO:87 and a light chain comprising the amino acid sequence SEQ        ID NO:73;    -   (xi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:88 and a light chain comprising the amino acid sequence SEQ        ID NO:73;    -   (xii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:82 and a light chain comprising the amino acid sequence SEQ        ID NO:74;    -   (xiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:83 and a light chain comprising the amino acid sequence        SEQ ID NO:74;    -   (xiv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:89 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (xv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:81 and a light chain comprising the amino acid sequence SEQ        ID NO:74;    -   (xvi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:90 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xvii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:91 and a light chain comprising the amino acid sequence        SEQ ID NO:75;    -   (xviii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:92 and a light chain comprising the amino acid sequence        SEQ ID NO:75;    -   (xix) a heavy chain comprising the amino acid sequence of SEQ ID        NO:93 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xx) a heavy chain comprising the amino acid sequence of SEQ ID        NO:86 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xxi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:94 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (xxii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:91 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:92 and a light chain comprising the amino acid sequence        SEQ ID NO:31;    -   (xxiv) a heavy chain comprising the amino acid sequence of SEQ        ID NO:93 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:87 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (xxvi) a heavy chain comprising the amino acid sequence of SEQ        ID NO:88 and a light chain comprising the amino acid sequence        SEQ ID NO:69;    -   (xxvii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:95 and a light chain comprising the amino acid sequence        SEQ ID NO:76;    -   (xxviii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:96 and a light chain comprising the amino acid sequence        SEQ ID NO:77;    -   (xxix) a heavy chain comprising the amino acid sequence of SEQ        ID NO:97 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxx) a heavy chain comprising the amino acid sequence of SEQ ID        NO:98 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (xxxi) a heavy chain comprising the amino acid sequence of SEQ        ID NO:99 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxxii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:100 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxxiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:85 and a light chain comprising the amino acid sequence        SEQ ID NO:74;    -   (xxxiv) a heavy chain comprising the amino acid sequence of SEQ        ID NO:89 and a light chain comprising the amino acid sequence        SEQ ID NO:74;    -   (xxxv) a heavy chain comprising the amino acid sequence of SEQ        ID NO:103 and a light chain comprising the amino acid sequence        SEQ ID NO:102;    -   (xxxvi) a heavy chain comprising the amino acid sequence of SEQ        ID NO:105 and a light chain comprising the amino acid sequence        SEQ ID NO:104;        -   wherein the VH amino acid sequence is at least 90%, 95%,            97%, 98% or 99% identical thereto and the a VL amino acid            sequence is at least 90%, 95%, 97%, 98% or 99% identical            thereto.

Preferred embodiments of the anti-CD47 antibodies described herein, arealso characterized by combinations of properties which are not exhibitedby prior art anti-CD47 antibodies proposed for human therapeutic use.Accordingly, the preferred anti-CD47 antibodies described herein arecharacterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells; and

d. induces death of susceptible human tumor cells.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. induces death of susceptible human tumor cells; and

e. causes no agglutination of human red blood cells (hRBCs).

In yet another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. induces death of susceptible human tumor cells; and

e. causes reduced agglutination of human red blood cells (hRBCs).

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. specifically binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells

d. induces death of susceptible human tumor cells; and

e. has reduced hRBC binding.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. causes no agglutination of human red blood cells (hRBCs); and

e. does not bind to hRBCs.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. specifically binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. causes no agglutination of human red blood cells (hRBCs); and

e. has reduced hRBC binding.

In another preferred embodiment described herein, the monoclonalantibody, or antigen binding fragment thereof specifically also binds tonon-human primate CD47, wherein non-human primate may include, but isnot limited to, cynomolgus monkey, green monkey, rhesus monkey andsquirrel monkey.

In yet another preferred embodiment described herein, the monoclonalantibody, or antigen binding fragment thereof binds to human, non-humanprimate, mouse, rabbit, and rat CD47.

Various forms of the anti-CD47 mAbs disclosed are contemplated herein.For example, the anti-CD47 mAbs can be full length humanized antibodieswith human frameworks and constant regions of the isotypes, IgA, IgD,IgE, IgG, and IgM, more particularly, IgG1, IgG2, IgG3, IgG4, and insome cases with various mutations to alter Fc receptor function orprevent Fab arm exchangeor an antibody fragment, e.g., a F(ab′)2fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., asdisclosed herein.

The preferred embodiments of the disclosure provide pharmaceutical orveterinary compositions comprising one or more of the anti-CD47 mAbs orfragments disclosed herein, optionally chimeric or humanized forms, anda pharmaceutically acceptable carrier, diluent, or excipient.

Prior to the present disclosure, there was a need to identify anti-CD47mAbs that possess the functional profiles as described herein. Theanti-CD47 mAbs of the present disclosure exhibit distinct combinationsof properties, particularly combinations of properties that render themAbs particularly advantageous or suitable for use in human therapy,particularly in the prevention and/or treatment of solid andhematological cancers, ischemia-reperfusion injury, autoimmune and/orinflammatory diseases.

Further scope of the applicability of the present disclosure will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the disclosure, aregiven by way of illustration only since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The above and other aspects, features, and advantages of the presentdisclosure will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawing(s), allof which are given by way of illustration only, and are not limitativeof the present disclosure.

FIG. 1A. Binding of VLX4 Humanized mAbs to Human OV10 Cells Expressinghuman CD47. Binding of VLX4 humanized mAbs (VLX4hum_01 IgG1, VLX4hum_02IgG1, VLX4hum_01 IgG4 PE, and VLX4hum_02 IgG4 PE) to human CD47 wasdetermined using a OV10 cell line expressing human CD47 (OV10 hCD47)cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates andwere confluent at the time of assay. Various concentrations of mAbs wereadded to the cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 1B. Binding of VLX4 Humanized mAbs to Human OV10 Cells Expressinghuman CD47. Binding of VLX4 humanized mAbs (VLX4hum_06 IgG4 PE,VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, and VLX4hum_13 IgG4 PE) to humanCD47 was determined using an OV10 CD47 cell-based ELISA. OV10 hCD47cells were plated into 96 well plates and were confluent at the time ofassay. Various concentrations of VLX4 representative mAbs were added tothe cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 2A. Binding of VLX4 Humanized mAbs to Human RBCs (hRBCs). Bindingof VLX4 humanized mAbs (VLX4hum_01 IgG1, VLX4hum_02 IgG1, VLX4hum_01IgG4 PE, and VLX4hum_02 IgG4PE) to human CD47 was determined usingfreshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37° C.with various concentrations of VLX4 mAbs, washed and incubated for 1 hrwith FITC-labeled donkey anti-human antibody. Cells were washed andantibody binding measured using flow cytometry.

FIG. 2B. Binding of VLX4 Humanized mAbs to Human RBCs. Binding of VLX4humanized mAbs (VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, and VLX4hum_13IgG4 PE) to human CD47 was determined using freshly isolated hRBCs.hRBCs were incubated for 60 minutes at 37° C. with variousconcentrations of VLX4 mAbs, washed and incubated for 1 hr withFITC-labeled donkey anti-human antibody. Cells were washed and antibodybinding measured using flow cytometry.

FIG. 3A. Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX8 IgG4PE chimera (xi) or humanized mAbs (VLX8hum_01IgG4PE, VLX8hum_04 IgG4 PE, VLX8hum_07 IgG4 PE, and VLX8hum_09 IgG4 PE)to human CD47 was determined using an OV10 hCD47 cell-based ELISA. OV10hCD47 cells were plated into 96 well plates and were confluent at thetime of assay. Various concentrations of VLX8 representative mAbs wereadded to the cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 3B. Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human CD47 was determinedusing an OV10 hCD47 cell-based ELISA. OV10 hCD47 cells were plated into96 well plates and were confluent at the time of assay. Variousconcentrations of VLX8 representative mAbs were added to the cells for 1hr. Cells were washed and then incubated with HRP-labelled secondaryantibody for 1 hr followed by addition of peroxidase substrate.

FIG. 4A. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8IgG4PE xi or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE,VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE) to human CD47 was determinedusing freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C. with various concentrations of VLX8 mAbs, washed and incubated for 1hr with FITC-labeled donkey anti-human antibody. Cells were washed andantibody binding measured using flow cytometry.

FIG. 4B. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2,VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47 was determined usingfreshly isolated human RBCs. RBCs were incubated for 1 hr at 37° C. withvarious concentrations of VLX8 mAbs, washed and incubated for 1 hr withFITC-labeled donkey anti-human antibody. Cells were washed and antibodybinding measured using flow cytometry.

FIG. 5A. Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_01 IgG2, VLX9hum_02IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05 IgG2) to humanCD47 was determined using an OV10 human CD47 cell-based ELISA. OV10hCD47 cells were plated into 96 well plates and were confluent at thetime of assay. Various concentrations of mAbs were added to the cellsfor 1 hr. Cells were washed and then incubated with HRP-labelledsecondary antibody for 1 hr followed by addition of peroxidasesubstrate.

FIG. 5B. Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_06 IgG2, VLX9hum_07IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) to humanCD47 was determined using a OV10 hCD47 cell-based ELISA. OV10 hCD47cells were plated into 96 well plates and were confluent at the time ofassay. Various concentrations of mAbs were added to the cells for 1 hr.Cells were washed and then incubated with HRP-labelled secondaryantibody for 1 hr followed by addition of peroxidase substrate.

FIG. 6. Binding of VLX9 Humanized mAbs to Human RBCs. Binding of VLX9IgG2 xi or humanized mAbs to human CD47 was determined using freshlyisolated human hRBCs. RBCs were incubated for 60 minutes at 37° C. withvarious concentrations of VLX9 mAbs, washed and incubated for 1 hr withFITC-labeled donkey anti-human antibody. Cells were washed and antibodybinding measured using flow cytometry.

FIG. 7. VLX4, VLX8, and VLX9 Humanized mAbs Block SIRPα binding to CD47on Jurkat Cells. 1.5×10⁶ Jurkat cells were incubated with 5 μg/ml ofVLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4 PE, VLX4hum_07IgG4 PE, VLX8hum_10 IgG4 PE, VLX4hum_11 IgG4 PE, VLX9hum_03 IgG2,VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody in RPMIcontaining 10% media for 30 min at 37° C. An equal volume offluorescently labeled SIRPα-Fc fusion protein was added and incubatedfor an additional 30 min at 37° C. Cells were washed and binding wasassessed using flow cytometry.

FIG. 8. VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat T Cellsby Human Macrophages. Human macrophages were plated at a concentrationof 1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hrs. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml of theVLX4 chimeric mAbs were added to the macrophage cultures and incubatedat 37° C. for 2 hrs. Non-phagocytosed Jurkat cells were removed andmacrophage cultures were washed extensively. Macrophages weretrypsinized and stained for CD14. Flow cytometry was used to determinethe percentage of CD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 9A. VLX4 Humanized mAbs Increase Phagocytosis of Jurkat T Cells byHuman Macrophages. Human macrophages were plated at a concentration of1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hrs. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml ofantibody were added to the macrophage cultures and incubated at 37° C.for 2 hrs. Non-phagocytosed Jurkat T cells were removed and macrophagecultures were washed extensively. Macrophages were trypsinized andstained for CD14. Flow cytometry was used to determine the percentage ofCD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 9B. VLX4 Humanized mAbs Increase Phagocytosis of Jurkat T Cells byHuman Macrophages. Human macrophages were plated at a concentration of1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hrs. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml ofantibody were added to the macrophage cultures and incubated at 37° C.for 2 hrs. Non-phagocytosed Jurkat T cells were removed and macrophagecultures were washed extensively. Macrophages were trypsinized andstained for CD14. Flow cytometry was used to determine the percentage ofCD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 10A. VLX8 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat TCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1μg/ml of the VLX8 chimeric mAbs were added to the macrophage culturesand incubated at 37° C. for 2 hrs. Non-phagocytosed Jurkat cells wereremoved and macrophage cultures were washed extensively. Macrophageswere trypsinized and stained for CD14. Flow cytometry was used todetermine the percentage of CD14⁺/CFSE⁺ cells in the total CD14+population.

FIG. 10B. VLX8 Humanized mAbs Increase Phagocytosis of Jurkat Cells byHuman Macrophages. Human macrophages were plated at a concentration of1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hrs. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml ofantibody were added to the macrophage cultures and incubated at 37° C.for 2 hrs. Non-phagocytosed Jurkat T cells were removed and macrophagecultures were washed extensively. Macrophages were trypsinized andstained for CD14. Flow cytometry was used to determine the percentage ofCD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 11A VLX9 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat T Cellsby Human Macrophages. Human macrophages were plated at a concentrationof 1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hours. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml of theVLX9 chimeric mAbs were added to the macrophage cultures and incubatedat 37° C. for two hours. Non-phagocytosed Jurkat cells were removed andmacrophage cultures were washed extensively. Macrophages weretrypsinized and stained for CD14. Flow cytometry was used to determinethe percentage of CD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 11B. VLX9 Humanized mAbs Increase Phagocytosis of Jurkat T Cells byHuman Macrophages. Human macrophages were plated at a concentration of1×10⁴ cells per well in a 96 well plate and allowed to adhere for 24hours. 5×10⁴ CFSE (1 μM) labeled human Jurkat T cells and 1 μg/ml ofantibody were added to the macrophage cultures and incubated at 37° C.for two hours. Non-phagocytosed Jurkat cells were removed and macrophagecultures were washed extensively. Macrophages were trypsinized andstained for CD14. Flow cytometry was used to determine the percentage ofCD14⁺/CFSE⁺ cells in the total CD14+ population.

FIG. 12A. Induction of Cell Death in Human Jurkat T Cells by SolubleVLX4 Humanized mAbs. Jurkat T cells (1×10⁴) were incubated with 1 μg/mlVLX4 humanized mAbs in 1 ml of RPMI media for 24 hours at 37° C. Cellswere then stained for annexin V and the signal was detected by flowcytometry.

FIG. 12B. Induction of Cell Death in Human Jurkat T Cells by SolubleVLX4 Humanized mAbs. Jurkat T cells (1×10⁴) were incubated with 1 μg/mlVLX4 humanized mAbs in 1 ml of RPMI media for 24 hours at 37° C. Cellswere then stained for annexin V and the signal was detected by flowcytometry.

FIG. 13A. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8CD47 Chimeric mAbs. Jurkat T ALL cells (1×10⁴) were incubated with 1μg/ml VLX8 humanized mAbs in 1 ml of RPMI media for 24 hrs at 37° C.Cells were then stained for annexin V and the signal was detected byflow cytometry.

FIG. 13B. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Humanized mAbs. Jurkat T ALL cells (1×104) were incubated with 1 μg/mlVLX8 humanized mAbs in 1 ml of RPMI media for 24 hrs at 37° C. Cellswere then stained for annexin V and the signal was detected by flowcytometry.

FIG. 14A. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9Murine/Human Chimeric mAbs. 1×10⁴ Jurkat cells were incubated with 1μg/ml of the VLX9 CD47 chimeric mAbs in 0.1 ml of RPMI media for 24hours 37° C. Cells were then stained with annexin V and the signalanalyzed by flow cytometry.

FIG. 14B. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9Humanized mAbs. Jurkat T ALL cells (1×10⁴) were incubated with 1 μg/mlVLX9 humanized mAbs in 1 ml of RPMI media for 24 hours at 37° C. Cellswere then stained for annexin V and the signal was detected by flowcytometry. VLX9 IgG2 (xi) is a murine/human chimera.

FIG. 15A. Agglutination of hRBCs by VLX4 Humanized mAbs.Hemagglutination was assessed following incubation of hRBCs with variousconcentrations of humanized VLX4 mAbs (25 μg/mL-0.4 ng/mL). Blood wasdiluted (1:50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added toU-bottomed 96 well plates with equal volumes of the antibodies (75 μl)and incubated for 3 hrs at 37° C. and overnight at 4° C.

FIG. 15B. Agglutination of hRBCs by VLX8 Chimeric and Humanized mAbs.Hemagglutination was assessed following incubation of hRBCs with variousconcentrations of humanized VLX4 mAbs (25 μg/mL-0.4 ng/mL). Blood wasdiluted (1:50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added toU-bottomed 96 well plates with equal volumes of the antibodies (75 μl)and incubated for 3 hrs at 37° C. and overnight at 4° C.

FIG. 16A. Agglutination of Human RBCs by VLX9 Humanized mAbs.Hemagglutination was assessed following incubation of human RBCs withvarious concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9mAbs. Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA.RBCs were added to U-bottomed 96 well plates with equal volumes of theantibodies (75 μl) and incubated for 3 hrs at 37° C. and overnight at 4°C.

FIG. 16B. Agglutination of Human RBCs by VLX9 Humanized mAbs.Hemagglutination was assessed following incubation of human RBCs withvarious concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9mAbs. Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA.RBCs were added to U-bottomed 96 well plates with equal volumes of theantibodies (75 μl) and incubated for 3 hrs at 37° C. and overnight at 4°C.

FIG. 17. VLX4 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ (BD Biosciences; Bedford, Mass.)mixture containing a suspension of 5×10⁶ Raji tumor cells. Five daysfollowing inoculation, tumor volumes were measured and mice withpalpable tumor volumes of 31-74 mm³ were randomized into 8-10/group.VLX4hum_07 or PBS (control) administration was initiated at this time.Mice were treated with 5 mg/kg of antibody 5×/week for 4 weeks byintraperitoneal injection. Tumor volumes and body weights were recordedtwice weekly.

FIG. 18. VLX8 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ (BD Biosciences; Bedford, Mass.)mixture containing a suspension of 5×10⁶ Raji tumor cells. Five daysfollowing inoculation, tumor volumes were measured and mice withpalpable tumor volumes of 31-74 mm³ were randomized into 8-10/group.VLX8hum_10 or PBS (control) administration was initiated at this time.Mice were treated with 5 mg/kg of antibody 5×/week for 4 weeks byintraperitoneal injection. Tumor volumes and body weights were recordedtwice weekly.

FIG. 19. VLX9 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ (BD Biosciences; Bedford, Mass.)mixture containing a suspension of 5×10⁶ Raji tumor cells. Five daysfollowing inoculation, tumor volumes were measured and mice withpalpable tumor volumes of 31-74 mm³ were randomized into 8-10/group.VLX9hum_08 IgG2 or PBS (control) administration was initiated at thistime. Mice were treated with 5 mg/kg of antibody 5×/week for 4 weeks byintraperitoneal injection. Tumor volumes and body weights were recordedtwice weekly.

FIG. 20A. Hemoglobin Levels in Blood following Administration of aHumanized VLX9 mAb to Cynomolgus Monkeys by Intravenous Infusion.VLX9hum_08 IgG2 or vehicle were administered as a one hour intravenousinfusion a dose of 5 mg/kg on day 1 and a dose of 15 mg/kg on day 18.Hemoglobin levels were monitored throughout the study and normalized tocontrol values.

FIG. 20B. RBC Levels in Blood following Administration of Humanized VLX9mAbs to Cynomolgus Monkeys by Intravenous Infusion. VLX9hum_08 IgG2 orvehicle was administered as a one hour intraveneous infusion a dose of 5mg/kg on day 1 and a dose of 15 mg/kg on day 18. RBC levels weremonitored throughout the study and normalized to control values.

FIG. 21. Immunohistochemical Staining of CD47 in Human Tumor Tissue withAnti-Murine/Rabbit Chimeric mAb. CD47 was localized in human breastcancer tissue using VLX4 mouse/rabbit chimeric mAb. Paraffin-embeddedtissue was sectioned, stained with 4 ug/ml of purified antibody andlocalized with anti-rabbit HRP secondary antibody. Arrows denotepositive areas of CD47 staining.

FIG. 22. Summary of Anti-CD47 Antibody Properties.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE DISCLOSURE Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. As used herein, the term“CD47”, “integrin-associated protein (IAP)”, “ovarian cancer antigenOA3”, “Rh-related antigen” and “MERG” are synonymous and may be usedinterchangeably.

The term “anti-CD47 antibody” refer to an antibody of the disclosurewhich is intended for use as a therapeutic or diagnostic agent, andtherefore will typically possess the binding affinity required to beuseful as a therapeutic and/or diagnostic agent.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts” with or directed against is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at a much loweraffinity (K_(d)>10⁻⁶). Antibodies include but are not limited to,polyclonal, monoclonal, chimeric, Fab fragments, Fab′ fragments, F(ab′)2fragments, single chain Fv fragments, and one-armed antibodies.

As used herein, the term “monoclonal antibody” (mAb) as applied to thepresent antibody compounds refers to an antibody that is derived from asingle copy or clone including, for example, any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.mAbs of the present disclosure preferably exist in a homogeneous orsubstantially homogeneous population. Complete mAbs contain 2 heavychains and 2 light chains.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

As disclosed herein, “antibody compounds” refers to mAbs andantigen-binding fragments thereof. Additional antibody compoundsexhibiting similar functional properties according to the presentdisclosure can be generated by conventional methods. For example, micecan be immunized with human CD47 or fragments thereof, the resultingantibodies can be recovered and purified, and determination of whetherthey possess binding and functional properties similar to or the same asthe antibody compounds disclosed herein can be assessed by the methodsdisclosed in Examples 3-11, below. Antigen-binding fragments can also beprepared by conventional methods. Methods for producing and purifyingantibodies and antigen-binding fragments are well known in the art andcan be found, for example, in Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 5-8 and 15.

The monoclonal antibodies encompass antibodies in which a portion of theheavy and/or light chain is identical with, or homologous to,corresponding sequences in murine antibodies, in particular the murineCDRs, while the remainder of the chain(s) is (are) identical with, orhomologous to, corresponding sequences in human antibodies. Otherembodiments of the disclosure include antigen-binding fragments of thesemonoclonal antibodies that exhibit binding and biological propertiessimilar or identical to the monoclonal antibodies. The antibodies of thepresent disclosure can comprise kappa or lambda light chain constantregions, and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions,including those of IgG subclasses IgG1, IgG2, IgG3, and IgG4 and in somecases with various mutations to alter Fc receptor function.

The monoclonal antibodies containing the presently disclosed murine CDRscan be prepared by any of the various methods known to those skilled inthe art, including recombinant DNA methods.

Reviews of current methods for antibody engineering and improvement canbe found, for example, in P. Chames, Ed., (2012) Antibody Engineering:Methods and Protocols, Second Edition (Methods in Molecular Biology,Book 907), Humana Press, ISBN-10: 1617799734; C. R. Wood, Ed., (2011)Antibody Drug Discovery (Molecular Medicine and Medicinal Chemistry,Book 4), Imperial College Press; R. Kontermann and S. Dubel, Eds.,(2010) Antibody Engineering Volumes 1 and 2 (Springer Protocols), SecondEdition; and W. Strohl and L. Strohl (2012) Therapeutic antibodyengineering: Current and future advances driving the strongest growtharea in the pharmaceutical industry, Woodhead Publishing.

Methods for producing and purifying antibodies and antigen-bindingfragments are well known in the art and can be found, for example, inHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15.

A full-length antibody as it exists naturally is a “Y” shapedimmunoglobulin (Ig) molecule comprising four polypeptide chains: twoidentical heavy (H) chains and two identical light (L) chains,interconnected by disulfide bonds. The amino terminal portion of eachchain, termed the fragment antigen binding region (FAB), includes avariable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition via the complementarity determiningregions (CDRs) contained therein. The carboxy-terminal portion of eachchain defines a constant region (the “Fc” region) primarily responsiblefor effector function.

The CDRs are interspersed with regions that are more conserved, termedframeworks (“FRs”). Amino acid sequences of many FRs are well known inthe art. Each light chain variable region (LCVR) and heavy chainvariable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred toas “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain arereferred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of theresidues which form specific interactions with the antigen. Thenumbering and positioning of CDR amino acid residues within the LCVR andHCVR regions are in accordance with the well-known Kabat numberingconvention Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242.

As described herein, the “antigen-binding site” can also be defined asthe “Hypervariable regions”, “HVRs”, or “HVs”, and refer to thestructurally hypervariable regions of antibody variable domains asdefined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917,1987). There are six HVRs, three in VH (H1, H2, H3) and three in VL (L1,L2, L3). We used herein CDRs as defined by Kabat except in H-CDR1, whichis extended to include H1.

There are five types of mammalian immunoglobulin (Ig) heavy chains,denoted by the Greek letters α (alpha), δ (delta), ε (epsilon), γ(gamma), and μ (mu), which define the class or isotype of an antibody asIgA, IgD, IgE, IgG, or IgM, respectively. IgG antibodies can be furtherdivided into subclasses, for example, IgG1, IgG2, IgG3, and IgG4.

Each heavy chain type is characterized by a particular constant regionwith a sequence well known in the art. The constant region is identicalin all antibodies of the same isotype, but differs in antibodies ofdifferent isotypes. Heavy chains γ, α, and δ have a constant regioncomposed of three tandem immunoglobulin (Ig) domains, and a hinge regionfor added flexibility. Heavy chains μ and c have a constant regioncomposed of four Ig domains.

The hinge region is a flexible amino acid stretch that links the Fc andFab portions of an antibody. This regions contains cysteine residuesthat can form disulfide bonds, connecting two heavy chains together.

The variable region of the heavy chain differs in antibodies produced bydifferent B cells, but is the same for all antibodies produced by asingle B cell or B cell clone. The variable region of each heavy chainis approximately 110 amino acids long and is composed of a single Igdomain.

In mammals, light chains are classified as kappa (κ) or lambda (k), andare characterized by a particular constant region as known in the art. Alight chain has two successive domains: one variable domain at theamino-terminal end, and one constant domain at the carboxy-terminal end.

Each antibody contains two light chains that are always identical; onlyone type of light chain, κ or k, is present per antibody in mammals.

The Fc region, composed of two heavy chains that contribute three orfour constant domains depending on the class of the antibody, plays arole in modulating immune cell activity. By binding to specificproteins, the Fc region ensures that each antibody generates anappropriate immune response for a given antigen. The Fc region alsobinds to various cell receptors, such as Fc receptors, and other immunemolecules, such as complement proteins. By doing this, it mediatesdifferent physiological effects, including opsonization, cell lysis, anddegranulation of mast cells, basophils and eosinophils.

As used herein, the term “epitope” refers to a specific arrangement ofamino acids located on a peptide or protein to which an antibody orantibody fragment binds. Epitopes often consist of a chemically activesurface grouping of molecules such as amino acids or sugar side chains,and have specific three dimensional structural characteristics as wellas specific charge characteristics. Epitopes can be linear, i.e.,involving binding to a single sequence of amino acids, orconformational, i.e., involving binding to two or more sequences ofamino acids in various regions of the antigen that may not necessarilybe contiguous in the linear sequence.

As used herein, the terms “specifically binds”, “bind specifically”,“specific binding”, and the like as applied to the present antibodycompounds refer to the ability of a specific binding agent (such as anantibody) to bind to a target molecular species in preference to bindingto other molecular species with which the specific binding agent andtarget molecular species are admixed. A specific binding agent is saidspecifically to recognize a target molecular species when it can bindspecifically to that target.

As used herein, the term “binding affinity” refers to the strength ofbinding of one molecule to another at a site on the molecule. If aparticular molecule will bind to or specifically associate with anotherparticular molecule, these two molecules are said to exhibit bindingaffinity for each other. Binding affinity is related to the associationconstant and dissociation constant for a pair of molecules, but it isnot critical to the methods herein that these constants be measured ordetermined. Rather, affinities as used herein to describe interactionsbetween molecules of the described methods are generally apparentaffinities (unless otherwise specified) observed in empirical studies,which can be used to compare the relative strength with which onemolecule (e.g., an antibody or other specific binding partner) will bindtwo other molecules (e.g., two versions or variants of a peptide). Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

As used herein, the term “sequence identity” means the percentage ofidentical nucleotide or amino acid residues at corresponding positionsin two or more sequences when the sequences are aligned to maximizesequence matching, i.e., taking into account gaps and insertions.Identity can be readily calculated by known methods, including but notlimited to those described in: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Methods to determine identity aredesigned to give the largest match between the sequences tested.Moreover, methods to determine identity are codified in publiclyavailable computer programs.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, by thehomology alignment algorithms, by the search for similarity method or,by computerized implementations of these algorithms (GAP, BESTFIT,PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys,Inc., San Diego, Calif., United States of America), or by visualinspection. See generally, Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402(1997).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;and Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold.

These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always; 0) and N (penalty scorefor mismatching residues; always; 0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a word length (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a word length (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is in one embodiment less than about0.1, in another embodiment less than about 0.01, and in still anotherembodiment less than about 0.001.

As used herein, the terms “humanized”, “humanization”, and the like,refer to grafting of the murine monoclonal antibody CDRs disclosedherein to human FRs and constant regions. Also encompassed by theseterms are possible further modifications to the murine CDRs, and humanFRs, by the methods disclosed in, for example, Kashmiri et al. (2005)Methods 36(1):25-34 and Hou et al. (2008) J. Biochem. 144(1):115-120,respectively, to improve various antibody properties, as discussedbelow.

As used herein, the term “humanized antibodies” refers to mAbs andantigen binding fragments thereof, including the antibody compoundsdisclosed herein, that have binding and functional properties accordingto the disclosure similar to those disclosed herein, and that have FRsand constant regions that are substantially human or fully humansurrounding CDRs derived from a non-human antibody.

As used herein, the term “FR” or “framework sequence” refers to any oneof FRs 1 to 4. Humanized antibodies and antigen binding fragmentsencompassed by the present disclosure include molecules wherein any oneor more of FRs 1 to 4 is substantially or fully human, i.e., wherein anyof the possible combinations of individual substantially or fully humanFRs 1 to 4, is present. For example, this includes molecules in whichFR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially orfully human. Substantially human frameworks are those that have at least80% sequence identity to a known human germline framework sequence.Preferably, the substantially human frameworks have at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequenceidentity, to a framework sequence disclosed herein, or to a known humangermline framework sequence.

Fully human frameworks are those that are identical to a known humangermline framework sequence. Human FR germline sequences can be obtainedfrom the international ImMunoGeneTics (IMGT) database and from TheImmunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc,Academic Press, 2001, the contents of which are herein incorporated byreference in their entirety.

The Immunoglobulin Facts Book is a compendium of the human germlineimmunoglobulin genes that are used to create the human antibodyrepertoire, and includes entries for 203 genes and 459 alleles, with atotal of 837 displayed sequences. The individual entries comprise allthe human immunoglobulin constant genes, and germline variable,diversity, and joining genes that have at least one functional or openreading frame allele, and which are localized in the three major loci.For example, germline light chain FRs can be selected from the groupconsisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27,IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16,IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39 andIGKV1D-33 and IGKJ1-5 and germline heavy chain FRs can be selected fromthe group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5,IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15,IGHV3-20, IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23,IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59 and IGHV5-51 and IGHJ1-6.

Substantially human FRs are those that have at least 80% sequenceidentity to a known human germline FR sequence. Preferably, thesubstantially human frameworks have at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity, to a framework sequences disclosed herein, or to a known humangermline framework sequence.

CDRs encompassed by the present disclosure include not only thosespecifically disclosed herein, but also CDR sequences having sequenceidentities of at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto a CDR sequence disclosed herein. Alternatively, CDRs encompassed bythe present disclosure include not only those specifically disclosedherein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acidchanges at corresponding positions compared to CDR sequences disclosedherein. Such sequence identical, or amino acid modified, CDRs preferablybind to the antigen recognized by the intact antibody.

Humanized antibodies in addition to those disclosed herein exhibitingsimilar functional properties according to the present disclosure can begenerated using several different methods Almagro et al. Frontiers inBiosciences. Humanization of antibodies. (2008) Jan. 1; 13:1619-33. Inone approach, the parent antibody compound CDRs are grafted into a humanframework that has a high sequence identity with the parent antibodycompound framework. The sequence identity of the new framework willgenerally be at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identical to the sequence of thecorresponding framework in the parent antibody compound. In the case offrameworks having fewer than 100 amino acid residues, one, two, three,four, five, six, seven, eight, nine, or ten amino acid residues can bechanged. This grafting may result in a reduction in binding affinitycompared to that of the parent antibody. If this is the case, theframework can be back-mutated to the parent framework at certainpositions based on specific criteria disclosed by Queen et al. (1991)Proc. Natl. Acad. Sci. USA 88:2869. Additional references describingmethods useful to generate humanized variants based on homology and backmutations include as described in Olimpieri et al. Bioinformatics. 2015Feb. 1; 31(3):434-435 and U.S. Pat. Nos. 4,816,397, 5,225,539, and5,693,761; and the method of Winter and co-workers (Jones et al. (1986)Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; andVerhoeyen et al. (1988) Science 239:1534-1536.

Humanization began with chimerization, a method developed during thefirst half of the 1980's (Morrison, S. L., M. J. Johnson, L. A.Herzenberg & V. T. Oi: Chimeric human antibody molecules: mouseantigen-binding domains with human constant region domains. Proc. Natl.Acad. Sci. USA., 81, 6851-5 (1984)), consisting of combining thevariable (V) domains of murine antibodies with human constant (C)domains to generate molecules with ˜70% of human content.

Several different methods can be used to generate humanized antibodies,which are described herein. In one approach, the parent antibodycompound CDRs are grafted into a human FR that has a high sequenceidentity with the parent antibody compound framework. The sequenceidentity of the new FR will generally be at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to the sequence of the corresponding FR in theparent antibody compound. In the case of FRs having fewer than 100 aminoacid residues, one, two, three, four, five, or more amino acid residuescan be changed. This grafting may result in a reduction in bindingaffinity compared to that of the parent antibody. If this is the case,the FR can be back-mutated to the parent framework at certain positionsbased on specific criteria disclosed by Queen et al. (1991) Proc. Natl.Acad. Sci. USA 88:2869. Additional references describing methods usefulto generate humanized variants based on homology and back mutationsinclude as described in Olimpieri et al. Bioinformatics. 2015 Feb. 1;31(3):434-435 and U.S. Pat. Nos. 4,816,397, 5,225,539, and 5,693,761;and the method of Winter and co-workers (Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536.

The identification of residues to consider for back-mutation can becarried out as described below. When an amino acid falls under thefollowing category, the framework amino acid of the human germ-linesequence that is being used (the “acceptor FR”) is replaced by aframework amino acid from a framework of the parent antibody compound(the “donor FR”):

(a) the amino acid in the human FR of the acceptor framework is unusualfor human frameworks at that position, whereas the corresponding aminoacid in the donor immunoglobulin is typical for human frameworks at thatposition;

(b) the position of the amino acid is immediately adjacent to one of theCDRs; or

(c) any side chain atom of a framework amino acid is within about 5-6angstroms (center-to-center) of any atom of a CDR amino acid in a threedimensional immunoglobulin model.

When each of the amino acids in the human FR of the acceptor frameworkand a corresponding amino acid in the donor framework is generallyunusual for human frameworks at that position, such amino acid can bereplaced by an amino acid typical for human frameworks at that position.This back-mutation criterion enables one to recover the activity of theparent antibody compound.

Another approach to generating humanized antibodies exhibiting similarfunctional properties to the antibody compounds disclosed hereininvolves randomly mutating amino acids within the grafted CDRs withoutchanging the framework, and screening the resultant molecules forbinding affinity and other functional properties that are as good as, orbetter than, those of the parent antibody compounds. Single mutationscan also be introduced at each amino acid position within each CDR,followed by assessing the effects of such mutations on binding affinityand other functional properties. Single mutations producing improvedproperties can be combined to assess their effects in combination withone another.

Further, a combination of both of the foregoing approaches is possible.After CDR grafting, one can back-mutate specific FRs in addition tointroducing amino acid changes in the CDRs. This methodology isdescribed in Wu et al. (1999) J. Mol. Biol. 294: 151-162.

Applying the teachings of the present disclosure, a person skilled inthe art can use common techniques, e.g., site-directed mutagenesis, tosubstitute amino acids within the presently disclosed CDR and FRsequences and thereby generate further variable region amino acidsequences derived from the present sequences. Up to all naturallyoccurring amino acids can be introduced at a specific substitution site.The methods disclosed herein can then be used to screen these additionalvariable region amino acid sequences to identify sequences having theindicated in vivo functions. In this way, further sequences suitable forpreparing humanized antibodies and antigen-binding portions thereof inaccordance with the present disclosure can be identified. Preferably,amino acid substitution within the frameworks is restricted to one, two,three, four, or five positions within any one or more of the four lightchain and/or heavy chain FRs disclosed herein. Preferably, amino acidsubstitution within the CDRs is restricted to one, two, three, four, orfive positions within any one or more of the three light chain and/orheavy chain CDRs. Combinations of the various changes within these FRsand CDRs described above are also possible.

That the functional properties of the antibody compounds generated byintroducing the amino acid modifications discussed above conform tothose exhibited by the specific molecules disclosed herein can beconfirmed by the methods in Examples disclosed herein.

As described above, to circumvent the problem of eliciting humananti-murine antibody (HAMA) response in patients, murine antibodies havebeen genetically manipulated to progressively replace their murinecontent with the amino acid residues present in their human counterpartsby grafting their complementarity determining regions (CDRs) onto thevariable light (V_(L)) and variable heavy (V_(H)) frameworks of humanimmunoglobulin molecules, while retaining those murine frameworkresidues deemed essential for the integrity of the antigen-combiningsite. However, the xenogeneic CDRs of the humanized antibodies may evokeanti-idiotypic (anti-Id) response in patients.

To minimize the anti-Id response, a procedure to humanize xenogeneicantibodies by grafting onto the human frameworks only the CDR residuesmost crucial in the antibody-ligand interaction, called “SDR grafting”,has been developed, wherein only the crucial specificity determiningresidues (SDRs) of CDRS are grafted onto the human frameworks. Thisprocedure, described in Kashmiri et al. (2005) Methods 36(1):25-34,involves identification of SDRs through the help of a database of thethree-dimensional structures of the antigen-antibody complexes of knownstructures, or by mutational analysis of the antibody-combining site. Analternative approach to humanization involving retention of more CDRresidues is based on grafting of the ‘abbreviated’ CDRs, the stretchesof CDR residues that include all the SDRs. Kashmiri et al. alsodiscloses a procedure to assess the reactivity of humanized antibodiesto sera from patients who had been administered the murine antibody.

Another strategy for constructing human antibody variants with improvedimmunogenic properties is disclosed in Hou et al. (2008) J. Biochem.144(1):115-120. These authors developed a humanized antibody from 4C8, amurine anti-human CD34 monoclonal antibody, by CDR grafting using amolecular model of 4C8 built by computer-assisted homology modelling.Using this molecular model, the authors identified FR residues ofpotential importance in antigen binding. A humanized version of 4C8 wasgenerated by transferring these key murine FR residues onto a humanantibody framework that was selected based on homology to the murineantibody FR, together with the murine CDR residues. The resultinghumanized antibody was shown to possess antigen-binding affinity andspecificity similar to that of the original murine antibody, suggestingthat it might be an alternative to murine anti-CD34 antibodies routinelyused clinically.

Embodiments of the present disclosure encompass antibodies created toavoid recognition by the human immune system containing CDRs disclosedherein in any combinatorial form such that contemplated mAbs can containthe set of CDRs from a single murine mAb disclosed herein, or light andheavy chains containing sets of CDRs comprising individual CDRs derivedfrom two or three of the disclosed murine mAbs. Such mAbs can be createdby standard techniques of molecular biology and screened for desiredactivities using assays described herein. In this way, the disclosureprovides a “mix and match” approach to create novel mAbs comprising amixture of CDRs from the disclosed murine mAbs to achieve new, orimproved, therapeutic activities.

Monoclonal antibodies or antigen-binding fragments thereof encompassedby the present disclosure that “compete” with the molecules disclosedherein are those that bind human CD47 at site(s) that are identical to,or overlapping with, the site(s) at which the present molecules bind.

Competing monoclonal antibodies or antigen-binding fragments thereof canbe identified, for example, via an antibody competition assay. Forexample, a sample of purified or partially purified human CD47extracellular domain can be bound to a solid support. Then, an antibodycompound, or antigen binding fragment thereof, of the present disclosureand a monoclonal antibody or antigen-binding fragment thereof suspectedof being able to compete with such disclosure antibody compound areadded. One of the two molecules is labeled. If the labeled compound andthe unlabeled compound bind to separate and discrete sites on CD47, thelabeled compound will bind to the same level whether or not thesuspected competing compound is present. However, if the sites ofinteraction are identical or overlapping, the unlabeled compound willcompete, and the amount of labeled compound bound to the antigen will belowered. If the unlabeled compound is present in excess, very little, ifany, labeled compound will bind. For purposes of the present disclosure,competing monoclonal antibodies or antigen-binding fragments thereof arethose that decrease the binding of the present antibody compounds toCD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99%. Details of procedures for carrying out such competitionassays are well known in the art and can be found, for example, inHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. Such assays can bemade quantitative by using purified antibodies. A standard curve isestablished by titrating one antibody against itself, i.e., the sameantibody is used for both the label and the competitor. The capacity ofan unlabeled competing monoclonal antibody or antigen-binding fragmentthereof to inhibit the binding of the labeled molecule to the plate istitrated. The results are plotted, and the concentrations necessary toachieve the desired degree of binding inhibition are compared.

Whether mAbs or antigen-binding fragments thereof that compete withantibody compounds of the present disclosure in such competition assayspossess the same or similar functional properties of the presentantibody compounds can be determined via these methods in conjunctionwith the methods described in Examples 3-5, below. In variousembodiments, competing antibodies for use in the therapeutic methodsencompassed herein possess biological activities as described herein inthe range of from about 50% to about 100% or about 125%, or more,compared to that of the antibody compounds disclosed herein. In someembodiments, competing antibodies possess about 50%, about 60%, about70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, oridentical biological activity compared to that of the antibody compoundsdisclosed herein as determined by the methods disclosed in the Examplespresented below.

The mAbs or antigen-binding fragments thereof, or competing antibodiesuseful in the compositions and methods can be any of the isotypesdescribed herein. Furthermore, any of these isotypes can comprisefurther amino acid modifications as follows.

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG1 isotype.

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to alter antibody half-life.

Antibody half-life is regulated in large part by Fc-dependentinteractions with the neonatal Fc receptor (Roopenian and Alikesh,2007). The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody can be modifiedto increase half-life include, but are not limited to amino acidmodifications N434A, T307A/E380A/N434A (Petkova et al., 2006, Yeung etal., 2009); M252Y/S254T/T256E (Dall'Acqua et al., 2006); T250Q/M428L(Hinton et al., 2006); and M428L/N434S (Zalev sky et al., 2010).

As opposed to increasing half-life, there are some circumstances wheredecreased half-life would be desired, such as to reduce the possibilityof adverse events associated with high Antibody-Dependent CellularCytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC)antibodies (Presta 2008). The human IgG1 constant region of themonoclonal antibody, antigen-binding fragment thereof, or competingantibody described herein can be modified to decrease half-life and/ordecrease endogenous IgG include, but are not limited to amino acidmodifications I253A (Petkova et al., 2006); P257I/N434H, D376V/N434H(Datta-Mannan et al., 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaroet al., 2005).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase or decrease antibody effector functions.These antibody effector functions include, but are not limited to,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), C1qbinding, and altered binding to Fc receptors.

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase antibody effector function include, but arenot limited to amino acid modifications S298A/E333A/K334 (Shields etal., 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006);F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, andF243L/R292P/Y300L/V3051/P396L (Stevenhagen et al., 2007); G236A,G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008);K326A/E333A, K326A/E333S and K326W/E333S (Idusogie et al., 2001); S267Eand S267E/L328F (Smith et al., 2012); H268F/S324T, S267E/H268F,S267E/S234T, and S267E/H268F/S324T (Moore et al., 2010); S298G/T299A(Sazinsky et al., 2008); E382V/M428I (Jung et al., 2010).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications N297A and N297Q (Bolt et al.,1993, Walker et al., 1989); L234A/L235A (Xu et al., 2000);K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaertet al., 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al., 2007);S267E/L328F (Chu et al., 2008).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications V234A/G237A (Cole et al., 1999);E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S,H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S,H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R,E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S,E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S,G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R,G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S,E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R,E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S,P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R,P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G,P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,P238D/E233D/G237D/H268Q/P271G/A330R,P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S(An et al., 2009, Mimoto, 2013).

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG2 isotype.

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase or decrease antibody effector functions.These antibody effector functions include, but are not limited to,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), andC1q binding, and altered binding to Fc receptors.

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase antibody effector function include, but arenot limited to the amino acid modification K326A/E333S (Idusogie et al.,2001).

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications V234A/G237A (Cole et al., 1999);E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S,H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S,H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R,E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S,E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S,G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R,G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S,E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R,E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S,P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R,P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G,P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,P238D/E233D/G237D/H268Q/P271G/A330R,P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S(An et al., 2009, Mimoto, 2013).

The Fc region of a human IgG2 of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to alter isoform and/or agonistic activity, include, butare not limited to amino acid modifications C127S (C_(H1) domain),C232S, C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightleet al., 2010).

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG3 isotype.

The human IgG3 constant region of the monoclonal antibody, or antigenbinding fragment thereof, wherein said human IgG3 constant region of themonoclonal antibody, or antigen-binding fragment thereof can be modifiedat one or more amino acid(s) to increase antibody half-life,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), or apoptosis activity.

The human IgG3 constant region of the monoclonal antibody, orantigen-binding fragment thereof, wherein said human IgG3 constantregion of the monoclonal antibody, or antigen-binding fragment thereofcan be modified at amino acid R435H to increase antibody half-life.

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG4 isotype.

The human IgG4 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector functions. These antibodyeffector functions include, but are not limited to, Antibody-DependentCellular Cytotoxicity (ADCC) and Antibody-Dependent CellularPhagocytosis (ADCP).

The human IgG4 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to prevent Fab arm exchange and/or decrease antibodyeffector function include, but are not limited to amino acidmodifications F234A/L235A (Alegre et al., 1994); S228P, L235E andS228P/L235E (Reddy et al., 2000).

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer”, “cancerous”, and “tumor” are not mutually exclusiveas used herein. The terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby aberrant cell growth/proliferation. Examples of cancers include, butare not limited to, carcinoma, lymphoma (i.e., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

The term “susceptible cancer” as used herein refers to a cancer, cellsof which express CD47, and are responsive to treatment with an antibodyor antigen binding fragment thereof, or competing antibody or antigenbinding fragment thereof, of the present disclosure.

The term “autoimmune disease” as used herein refers to when the body'simmune system turns against itself and mistakenly attacks healthy cells.

The term “inflammatory disease” as used herein refers to a diseasecharacterized by inflammation which is a fundamental pathologic processconsisting of a dynamic complex of histologically apparent cytologicchanges, cellular infiltration, and mediator release that occurs in theaffected blood vessels and adjacent tissues in response to an injury orabnormal stimulation caused by a physical, chemical, or biologic agent,including the local reactions and resulting morphologic changes; thedestruction or removal of the injurious material; and the responses thatlead to repair and healing.

The term “autoinflammatory disease” as used herein refers to a diseasethat results when the innate immune system causes inflammation forunknown reasons.

As used herein, the term “ischemia” refers to a vascular phenomenon inwhich a decrease in the blood supply to a bodily organ, tissue, or partis caused, for instance, by constriction or obstruction of one or moreblood vessels. Ischemia sometimes results from vasoconstriction orthrombosis or embolism. Ischemia can lead to direct ischemic injury,tissue damage due to cell death caused by reduced oxygen supply.Ischemia can occur acutely, as during surgery, or from trauma to tissueincurred in accidents, injuries and war settings, or following harvestof organs intended for subsequent transplantation, for example. It canalso occur sub-acutely, as found in atherosclerotic peripheral vasculardisease, where progressive narrowing of blood vessels leads toinadequate blood flow to tissues and organs. When a tissue is subjectedto ischemia, a sequence of chemical events is initiated that mayultimately lead to cellular dysfunction and necrosis. If ischemia isended by the restoration of blood flow, a second series of injuriousevents ensue, producing additional injury. Thus, whenever there is atransient decrease or interruption of blood flow in a subject, theresultant injury involves two components—the direct injury occurringduring the ischemic interval, and the indirect or reperfusion injurythat follows.

“Ischemic stroke” can be caused by several different kinds of diseases.The most common problem is narrowing of the arteries in the neck orhead. This is most often caused by atherosclerosis, or gradualcholesterol deposition. If the arteries become too narrow, blood cellsmay collect in them and form blood clots (thrombi). These blood clotscan block the artery where they are formed (thrombosis), or can dislodgeand become trapped in arteries closer to the brain (embolism). Cerebralstroke can occur when atherosclerotic plaque separates away partiallyfrom the vessel wall and occludes the flow of blood through the bloodvessel.

As used herein, the term “Reperfusion” refers to restoration of bloodflow to tissue that is ischemic, due to decrease in blood flow.Reperfusion is a procedure for treating infarction or other ischemia, byenabling viable ischemic tissue to recover, thus limiting furthernecrosis. However, reperfusion can itself further damage the ischemictissue, causing reperfusion injury. In addition to the immediate injurythat occurs during deprivation of blood flow, “ischemic/reperfusioninjury” involves tissue injury that occurs after blood flow is restored.Current understanding is that much of this injury is caused by chemicalproducts, free radicals, and active biological agents released by theischemic tissues.

“Nitric oxide (NO) donor, precursor, or nitric oxide generating topicalagent” refers to a compound or agent that either delivers NO, or thatcan be converted to NO through enzymatic or non-enzymatic processes.Examples include, but are not limited to, NO gas, isosorbide dinitrite,nitrite, nitroprus side, nitroglycerin, 3-Morpholinosydnonimine (SIN-1),S-nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO(DETA/NO), S-nitrosothiols, Bidil®, and arginine.

“Soluble guanylyl cyclase (sGC)” is the receptor for nitric oxide invascular smooth muscle. In the cardiovascular system, nitric oxide isendogenously generated by endothelial nitric oxide synthase fromL-arginine, and activates soluble guanylyl cyclase in adjacent vascularsmooth muscle cells to increase cGMP levels, inducing vascularrelaxation. Nitric oxide binds to the normally reduced heme moiety ofsoluble guanylyl cyclase, and increases the formation of cGMP from GTP,leading to a decrease in intracellular calcium, vasodilation, andanti-inflammatory effects. Oxidation of the heme iron on sGC decreasesresponsiveness of the enzyme to nitric oxide, and promotesvasoconstriction. The nitric oxide-sGC-cGMP pathway therefore plays animportant role in cardiovascular diseases. Nitrogen-containing compoundssuch as sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, andsodium nitroprusside have been shown to stimulate sGC, causing anincrease in cGMP, and vascular relaxation. In contrast to stimulators ofsGC, which bind to reduced sGC, activators of sGC activate the oxidizedor heme-deficient sGC enzyme that is not responsive to nitric oxide,i.e., they stimulate sGC independent of redox state. While stimulatorsof of sGC can enhance the sensitivity of reduced sGC to nitric oxide,activators of sGC can increase sGC enzyme activity even when the enzymeis oxidized and is therefore less, or unresponsive, to nitric oxide.Thus, sGC activators are non-nitric oxide based. Note the reviews ofNossaman et al. (2012) Critical Care Research and Practice, Volume 2012,article 290805, and Derbyshire and Marletta (2012) Ann. Rev. Biochem.81:533-559.

“An agent that activates soluble guanylyl cyclase” refers, for example,to organic nitrates (Artz et al. (2002) J. Biol. Chem. 277:18253-18256);protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci. USA79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226-4233); BAY 41-2272and BAY 41-8543 (Stasch et al. (2001 Nature 410 (6825): 212-5),CMF-1571, and A-350619 (reviewed in Evgenov et al. (2006) Nat. Rev.Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Frey et al. (2008)Journal of Clinical Pharmacology 48 (12): 1400-10); BAY 63-2521(Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65).Additional soluble guanylyl cyclase activators are disclosed in Staschet al. (2011) Circulation 123:2263-2273; Derbyshire and Marletta (2012)Ann. Rev. Biochem. 81:533-559, and Nossaman et al. (2012) Critical CareResearch and Practice, Volume 2012, Article ID 290805, pages 1-12.

cGMP can also be increased by inhibiting degradation usingphosphodiesterase inhibitors. Examples of “an agent that inhibits cyclicnucleotide phosphodiesterases” include, tadalafil, vardenafil, udenafil,and sildenafil avanafil.

As used herein, term “treating” or “treat” or “treatment” means slowing,interrupting, arresting, controlling, stopping, reducing, or reversingthe progression or severity of a sign, symptom, disorder, condition, ordisease, but does not necessarily involve a total elimination of alldisease-related signs, symptoms, conditions, or disorders. The term“treating” and the like refer to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop.

As used herein, term “effective amount” refers to the amount or dose ofan antibody compound of the present disclosure which, upon single ormultiple dose administration to a patient or organ, provides the desiredtreatment or prevention.

The precise effective amount for any particular subject will depend upontheir size and health, the nature and extent of their condition, and thetherapeutics or combination of therapeutics selected for administration.The effective amount for a given patient is determined by routineexperimentation and is within the judgment of a clinician.Therapeutically effective amounts of the present antibody compounds canalso comprise an amount in the range of from about 0.1 mg/kg to about150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kgto about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per singledose administered to a harvested organ or to a patient. Knownantibody-based pharmaceuticals provide guidance in this respect. Forexample, Herceptin™ is administered by intravenous infusion of a 21mg/ml solution, with an initial loading dose of 4 mg/kg body weight anda weekly maintenance dose of 2 mg/kg body weight; Rituxan™ isadministered weekly at 375 mg/m²; for example.

A therapeutically effective amount for any individual patient can bedetermined by the health care provider by monitoring the effect of theantibody compounds on tumor regression, circulating tumor cells, tumorstem cells or anti-tumor responses. Analysis of the data obtained bythese methods permits modification of the treatment regimen duringtherapy so that optimal amounts of antibody compounds of the presentdisclosure, whether employed alone or in combination with one another,or in combination with another therapeutic agent, or both, areadministered, and so that the duration of treatment can be determined aswell. In this way, the dosing/treatment regimen can be modified over thecourse of therapy so that the lowest amounts of antibody compounds usedalone or in combination that exhibit satisfactory efficacy areadministered, and so that administration of such compounds is continuedonly so long as is necessary to successfully treat the patient. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g., whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

In some embodiments antibody compounds of the present disclosure can beused as medicaments in human and veterinary medicine, administered by avariety of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intratumoral, intranasal, enteral, sublingual,intravaginal, intravesiciular or rectal routes. The compositions canalso be administered directly into a lesion such as a tumor. Dosagetreatment may be a single dose schedule or a multiple dose schedule.Hypo sprays may also be used to administer the pharmaceuticalcompositions. Typically, the therapeutic compositions can be prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Veterinary applications include thetreatment of companion/pet animals, such as cats and dogs; workinganimals, such as guide or service dogs, and horses; sport animals, suchas horses and dogs; zoo animals, such as primates, cats such as lionsand tigers, bears, etc.; and other valuable animals kept in captivity.

Such pharmaceutical compositions can be prepared by methods well knownin the art. See, e.g., Remington: The Science and Practice of Pharmacy,21^(st) Edition (2005), Lippincott Williams & Wilkins, Philadelphia,Pa., and comprise one or more antibody compounds disclosed herein, and apharmaceutically or veterinarily acceptable, for example,physiologically acceptable, carrier, diluent, or excipient.

The present disclosure describes anti-CD47 mAbs with distinct functionalprofiles. These antibodies possess distinct combinations of propertiesselected from the following: 1) exhibit cross-reactivity with one ormore species homologs of CD47; 2) block the interaction between CD47 andits ligand SIRPα; 3) increase phagocytosis of human tumor cells, 4)induce death of susceptible human tumor cells; 5) do not induce celldeath of human tumor cells; 6) have reduced binding to human red bloodcells (hRBCs); 7) have no detectable binding to hRBCs; 8) cause reducedagglutination of hRBCs; 9) cause no detectable agglutination of hRBCs;10) reverse TSP1 inhibition of the nitric oxide (NO) pathway and/or 11)do not reverse TSP1 inhibition of the NO pathway.

The anti-CD47 antibodies and antigen binding fragments thereof of thepresent disclosure possess combinations of properties that are distinctfrom the anti-CD47 antibodies of the prior art. These properties andcharacteristics will now be described in further detail.

Binding to CD47 of Different Species

The anti-CD47 antibodies, and antigen binding fragments thereof, of thepresent disclosure bind human CD47. In certain embodiments, theanti-CD47 antibodies exhibit cross-reactivity with one or more specieshomologs of CD47, for example CD47 homologs of non-human primate origin.In certain embodiments, the anti-CD47 antibodies and antigen bindingfragments thereof of the present disclosure bind to human CD47 and toCD47 of non-human primate, mouse, rat, and/or rabbit origin. Thecross-reactivity with other species homologs can be particularlyadvantageous in the development and testing of therapeutic antibodies.For example, pre-clinical toxicology testing of therapeutic antibodiesis frequently carried out in non-human primate species including, butnot limited to, cynomolgus monkey, green monkey, rhesus monkey andsquirrel monkey. Cross-reactivity with these species homologs cantherefore be particularly advantageous for the development of antibodiesas clinical candidates.

Blocking the Interaction Between CD47 and SIRPα and PromotingPhagocytosis

CD47, also known as integrin associated protein (IAP), is a 50 kDa cellsurface receptor that is comprised of an extracellular N-terminal IgVdomain, a five membrane spanning transmembrane domain, and a shortC-terminal intracellular tail that is alternatively spliced.

Two ligands bind to CD47: Signal Regulatory Protein alpha (SIRPα) andThrombospondin-1 (TSP1). TSP1 is present in plasma and synthesized bymany cells, including platelets. SIRPα is expressed on hematopoieticcells, which include macrophages and dendritic cells.

When SIRPα on a phagocyte engages CD47 on a target cell, thisinteraction prevents phagocytosis of the target cell. The interaction ofCD47 and SIRPα effectively sends a “don't eat me” signal to thephagocyte (Oldenborg et al. Science 288: 2051-2054, 2000). Blocking theinteraction of SIRPα and CD47 with an anti-CD47 mAb in a therapeuticcontext can provide an effective anti-cancer treatment by promoting theuptake and clearance of cancer cells by the host's immune system. Thus,an important functional characteristic of some anti-CD47 mAbs is theability to block the interaction of CD47 and SIRPα, resulting inphagocytosis of CD47 expressing tumor cells by macrophages. Severalanti-CD47 mAbs have been shown to block the interaction of CD47 andSIRPα, including B6H12 (Seiffert et al. Blood 94:3633-3643, 1999; Latouret al. J. Immunol. 167: 2547-2554, 2001; Subramanian et al. Blood 107:2548-2556, 2006; Liu et al. J Biol. Chem. 277: 10028-10036, 2002; Rebreset al et al. J. Cellular Physiol. 205: 182-193, 2005), BRIC126(Vernon-Wilson et al. Eur J Immunol. 30: 2130-2137, 2000; Subramanian etal. Blood 107: 2548-2556, 2006), CC2C6 (Seiffert et al. Blood94:3633-3643, 1999), and 1F7 (Rebres et al. J. Cellular Physiol. 205:182-193, 2005). B6H12 and BRIC126 have also been shown to causephagocytosis of human tumor cells by human and mouse macrophages(Willingham et al. Proc Natl Acad Sci USA 109(17):6662-6667, 2012; Chaoet al. Cell 142:699-713, 2012; EP 2 242 512 B1). Other existinganti-CD47 mAbs, such as 2D3, does not block the interaction of CD47 andSIRPα (Seiffert et al. Blood 94:3633-3643, 1999; Latour et al. J.Immunol. 167: 2547-2554, 2001; Rebres et al. J. Cellular Physiol. 205:182-193, 2005), and does not cause phagocytosis of tumor cells(Willingham et al. Proc Natl Acad Sci USA 109(17):6662-6667, 2012; Chaoet al. Cell 142:699-713, 2012; EP 2 242 512 B1).

As used herein, the term “blocks SIRPα binding to human CD47” refers toa greater than 50% reduction of SIRPα-Fc binding to CD47 on Jurkat cellsby an anti-CD47 mAb.

The anti-CD47 mAbs of the disclosure described herein, block theinteraction of CD47 and SIRPα and increase phagocytosis of human tumorcells.

“Phagocytosis” of cancer cells refers to the engulfment and digestion ofsuch cells by macrophages, and the eventual digestion or degradation ofthese cancer cells and the release of digested or degraded cellularcomponents extracellularly, or intracellularly to undergo furtherprocessing. Anti-CD47 monoclonal antibodies that block SIRPα binding toCD47 increase macrophage phagocytosis of cancer cells. SIRPα binding toCD47 on cancer cells would otherwise allow these cells to escapemacrophage phagocytosis. The cancer cell may be viable or living cancercells.

Inducing Death of Tumor Cells

Some soluble anti-CD47 mAbs initiate a cell death program on binding toCD47 on tumor cells, resulting in collapse of mitochrondrial membranepotential, loss of ATP generating capacity, increased cell surfaceexpression of phosphatidylserine (detected by increased staining forannexin V) and cell death without the participation of caspases orfragmentation of DNA. Such soluble anti-CD47 mAbs have the potential totreat a variety of solid and hematological cancers. Several solubleanti-CD47 mAbs which have been shown to induce tumor cell death,including MABL-1, MABL-2 and fragments thereof (U.S. Pat. No. 8,101,719;Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem BiophysRes. Commun. 315: 912-8, 2004), Ad22 (Pettersen et al. J. Immuno. 166:4931-4942, 2001; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003), and1F7 (Manna et al. J. Immunol. 170: 3544-3553, 2003; Manna et al. CancerResearch, 64: 1026-1036, 2004). Some of the anti-CD47 mAbs of thedisclosure described herein induce cell death of human tumor cells.

The terms “inducing cell death” or “kills” and the like, are usedinterchangeably herein to mean that addition of an antibody compound ofthe present disclosure to cultured cancer cells causes these cells todisplay quantifiable characteristics associated with cell deathincluding any one, or more, of the following:

1. Increased binding of Annexin V (in the presence of calcium ion) tothe tumor cells as detected by flow cytometry or confocal fluorescencemicroscopy;

2. Increased uptake of the fluorescent compound propidium iodide (asassayed by flow cytometry) or 7-aminoactinomycin D (7-AAD as assayed byflow cytometry) or trypan blue (scored with light microscopy) by thetumor cells

3. Loss of mitochondrial function and membrane potential by the tumorcells as assayed by one of several available measures (potentiometricfluorescent dyes such as DiO-C6 or JC1 or formazan-based assays such asMTT or WST-1).

Induction of cell death refers to the ability of certain of the solubleanti-CD47 antibodies, murine antibodies, chimeric antibodies, humanizedantibodies, or antigen-binding fragments thereof (and competingantibodies and antigen-binding fragments thereof) disclosed herein tokill cancer cells via a cell autonomous mechanism without participationof complement or other cells including, but not limited to, T cells,neutrophils, natural killer cells, macrophages, or dendritic cells.Quantifiably, induction of cell death includes, but is not limited to, agreater than 2-fold increase in annexin V staining of human tumor cellscaused by soluble anti-CD47 mAb compared to the background obtained withthe negative control antibody (humanized, isotype-matched antibody).

Among the present humanized or chimeric mAbs, those that induce celldeath of human tumor cells cause increased Annexin V binding similar tothe findings reported for anti-CD47 mAbs Ad22 (Pettersen et al. J.Immuno. 166: 4931-4942, 2001; Lamy et al. J. Biol. Chem. 278:23915-23921, 2003); 1F7 (Manna and Frazier J. Immunol. 170:3544-3553,2003; Manna and Frazier Cancer Res. 64:1026-1036, 2004); and MABL-1 and2 (U.S. Pat. No. 7,531,643 B2; U.S. Pat. No. 7,696,325 B2; U.S. Pat. No.8,101,719 B2).

Cell viability assays are described in NCI/NIH guidance manual thatdescribes numerous types of cell based assays that can be used to assessinduction of cell death caused by CD47 antibodies: “Cell ViabilityAssays”, Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS,Helene A Benink, PhD, Tracy J Worzella, MS, and Lisa Minor, PhD.Contributor Information, published May 1, 2013.

Binding to hRBCs

CD47 is expressed on human erythrocytes (hRBCs) (Brown. J Cell Biol.111: 2785-2794, 1990; Avent. Biochem J., (1988) 251: 499-505; Knapp.Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira et al. Biochimica etBiophysica Acta 1818: 481-490, 2012; Petrova P. et al. Cancer Res 2015;75(15 Suppl): Abstract nr 4271). It has been shown that anti-CD47 mAbsbind to RBCs, including B6H12 (Brown et al. J. Cell Biol., 1990,Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012,Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271),BRIC125 (Avent. Biochem J., (1988) 251: 499-505), BRIC126 (Avent.Biochem J., (1988) 251: 499-505; Petrova P. et al. Cancer Res 2015;75(15 Suppl): Abstract nr 4271), 5F9 (Uger R. et al. Cancer Res 2014;74(19 Suppl): Abstract nr 5011, Liu et al. PLoS One. 2015 Sep. 21;10(9): e0137345; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract3019)), anti-CD47 antibodies disclosed in US Patent Publication2014/0161799, WO Publication 2014/093678, US Patent Publication2014/0363442, and CC2C6 (Petrova P. et al. Cancer Res 2015; 75(15Suppl): Abstract nr 4271, Uger R. et al. Cancer Res 2014; 74(19 Suppl):Abstract nr 5011). It has also been shown that a SIRPα-Fc fusionprotein, which binds to human CD47, has reduced binding to human RBCscompared to other human cells (Uger R. et al. Cancer Res 2014; 74(19Suppl): Abstract nr 5011). Binding to RBCs can be reduced by generationof bi-specific antibodies with only one CD47 binding arm (Masternak etal. Cancer Res 2015; 75(15 Suppl): Abstract nr 2482).

Because some anti-CD47 mAbs have been shown to result in reduction ofRBCs when administered to cynomolgus monkeys (Mounho-Zamora B. et al.The Toxicologist, Supplement to Toxicological Sciences, 2015; 144 (1):Abstract 596: 127, Liu et al. PLoS One. 2015 Sep. 21; 10(9): e0137345;Pietsch et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 2470), it ishighly desirable to identify anti-CD47 mAbs that do not bind toCD47-expressing RBCs.

As used herein, the terms “red blood cell(s)” and “erythrocyte(s)” aresynonymous and used interchangeably herein.

As used herein, the terms “reduced binding to hRBCs”, refers to theK_(d) of an anti-CD47 mAb binding to a hRBC which is 10-fold or greaterthan the K_(d) on a human tumor cell, wherein the tumor cell is anOV10hCD47 cell.

As used herein, the term “no binding” or “NB”, refers to no measurablebinding to hRBCs at an anti-CD47 mAb concentration up to and including100 μg/ml.

Prior to the disclosure described herein, no anti-CD47 mAbs have beenreported that do not bind to human RBCs expressing CD47.

Some of the anti-CD47 mAbs, disclosed herein, have reduced or nodetectable binding to human RBCs.

Agglutination of RBCs

Red blood cell (RBC) agglutination or hemagglutination is a homotypicinteraction that occurs when RBCs aggregate or clump together followingincubation with various agents, including antibodies to RBC antigens andcell surface proteins such as CD47. Many anti-CD47 antibodies have beenreported to cause hemagglutination of isolated human RBCs in vitro, in aconcentration dependent manner, including B6H12, BRIC126, MABL-1,MABL-2, CC2C6, and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl):Abstract nr 5011, U.S. Pat. No. 9,045,541, Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract 3019)).This functional effect requires binding to RBCs by an intact, bivalentantibody and can be reduced or eliminated by generating antibodyfragments, either a F(ab′) or svFv (Uno et al. Oncol Rep. 17: 1189-94,2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004) orbi-specific antibodies with only one CD47 binding arm (Masternak et al.Cancer Res 2015; 75(15 Suppl): Abstract nr 2482). Other functionalproperties of these fragments, including cell killing, were shown to beeither reduced or retained in these fragments (Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004). The mouse antibody 2D3 is an example of an anti-CD47 antibodythat binds to CD47 on red blood cells but does not causehemagglutination (U.S. Pat. No. 9,045,541, Petrova et al. Cancer Res2015; 75(15 Suppl): Abstract nr 4271). Hemagglutination has been shownto be reduced/eliminated by reducing the binding selectively to humanRBCs, but not other cells, using a SIRPα-Fc fusion protein (Uger R. etal. Blood 2013; 122(21): 3935). In addition, mouse anti-CD47 mAb 2A1 andhumanized versions of 2A1 have been reported to block CD47/SIRPα but donot exhibit hemagglutination activity (U.S. Pat. No. 9,045,541). A smallnumber of a panel of mouse anti-human CD47 antibodies (3 of 23) werereported to not cause hemagglutination of human RBCs (Pietsch E et al.Cancer Res 2015; 75(15 Suppl): Abstract nr 2470). Therefore, prior tothe disclosure described herein, there was a need to identify CD47 mAbsthat block SIRPα/CD47 binding, have no detectable or reduced binding toRBCs and/or cause no hemagglutination. The term “agglutination” refersto cellular clumping, while the term “hemagglutination” refers toclumping of a specific subset of cells, i.e., RBCs. Thus,hemagglutination is a type of agglutination.

As used herein, the term “reduced hemagglutination” refers to measurableagglutination activity of hRBCs at anti-CD47 mAb concentrations greaterthat 1.85 μg/ml, and no measurable activity at concentrations less thanor equal to 1.85 μg/ml.

As used herein, the term “no detectable hemagglutination”, refers to nomeasurable agglutination activity of hRBCs at anti-CD47 mAbconcentrations greater or equal to 0.3 pg/mL to a concentration lessthan or equal to 50 μg/mL.

Some of the anti-CD47 antibodies described herein, cause reduced or nodetectable hemagglutination of human RBCs.

Modulation of the NO Pathway

As noted above, TSP1 is also a ligand for CD47. The TSP1/CD47 pathwayopposes the beneficial effects of the NO pathway in many cell types,including, but not limited to, vascular cells. The NO pathway consistsof any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOSIII) that generate bioactive gas NO using arginine as a substrate. NOcan act within the cell in which it is produced, or in neighboringcells, to activate the enzyme soluble guanylyl cyclase that produces themessenger molecule cyclic GMP (cGMP). The proper functioning of theNO/cGMP pathway is essential for protecting the cardiovascular systemagainst stresses including, but not limited to, those resulting fromwounding, inflammation, hypertension, metabolic syndrome, ischemia, andIRI. In the context of these cellular stresses the inhibition of theNO/cGMP pathway by the TSP1/CD47 system exacerbates the effects ofstress. This is a particular problem in the cardiovascular system whereboth cGMP and cAMP play important protective roles. There are many casesin which ischemia and reperfusion injury cause or contribute to disease,trauma, and poor outcomes of surgical procedures.

As disclosed herein, one of more of the chimeric or humanized anti-CD47antibodies will reverse TSP1 inhibition of cGMP production. Reversalwill be complete (>80%) or intermediate (20%-80%). This reversal of TSP1inhibition of cGMP production will demonstrate that the anti-CD47 mAbshave the ability to increase NO signaling and suggest utility inprotecting the cardiovascular system against stresses including, but notlimited to, those resulting from wounding, inflammation, hypertension,metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI).Additional assay systems, for example smooth muscle cell contraction,will also be expected to show that some of the chimeric or humanizedantibodies reverse the inhibitory actions of TSP1 on downstream effectsresulting from the activation of NO signaling.

As disclosed herein, “complete reversal of NO pathway inhibition” refersto greater than 80% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody.

As disclosed herein, “intermediate reversal of NO pathway inhibition”refers to 20-80% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody.

As disclosed herein, “no reversal of NO pathway inhibition” refers toless than 20% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody.

Preferred Combinations of Functional Properties

Anti-CD47 mAbs exist in the prior art with combinations of some, but notall, of the functional characteristics described herein. Previously, ithas been shown that humanized anti-CD47 mAbs such as AB6.12 IgG1,AB6.12-IgG4P, and AB6.12-IgG4PE (U.S. Pat. No. 9,045,541, US PatentPublication 2014/0161799, WO Publication 2014/093678, US PatentPublication 2014/0363442) and 5F9 (Mounho-Zamora B. et al. TheToxicologist, Supplement to Toxicological Sciences, 2015; 144 (1):Abstract 596: 127, Liu et al. PLoS One. 2015 Sep. 21; 10(9): e0137345)bind human CD47, block the interaction of CD47 and SIRPα and causephagocytosis of human tumor cells. The humanized CD47 mAbs AB6.12 IgG1,AB6.12-IgG4P, and AB6.12-IgG4PE also do not cause hemagglutination ofhuman RBCs (U.S. Pat. No. 9,045,541). The 5F9 humanized anti-CD47 mAbbinds to and causes hemagglutination of human RBCs (Uger R. et al.Cancer Res 2014; 74(19 Suppl): Abstract nr 5011, Sikic B. et al. J ClinOncol 2016; 34 (suppl; abstract 3019). Murine anti-CD47 mAbs B6H12,BRIC126, and CC2C6 block the interaction of CD47 and SIRPα, causephagocytosis, and bind to and cause hemagglutination of human RBCs(Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271,Seiffert et al. Blood 94:3633-3643, 1999; Vernon-Wilson et al. Eur JImmunol. 30: 2130-2137, 2000; Latour et al. J. Immunol. 167: 2547-2554,2001; Subramanian et al. Blood 107: 2548-2556, 2006; Liu et al. J Biol.Chem. 277: 10028-10036, 2002). Murine anti-CD47 mAbs MABL-1 and MABL-2bind to human CD47, induce tumor cell death and cause RBChemagglutination (U.S. Pat. No. 8,101,719); murine mAb Ad22 binds tohuman CD47 and induces tumor cell death (Pettersen et al. J. Immunol.166: 4931-4942, 2001; Lamy et al. J Biol Chem. 278: 23915-23921, 2003);and murine mAb 1F7 binds to human CD47, blocks the interaction of CD47and SIRPα and induces tumor cell death (Rebres et al. J. CellularPhysiol. 205: 182-193, 2005; Manna et al. J. Immunol. 170: 3544-3553,2003; Manna et al. Cancer Research, 64: 1026-1036, 2004).

Preferred embodiments of the anti-CD47 antibodies described herein, arealso characterized by combinations of properties which are not exhibitedby prior art anti-CD47 antibodies proposed for human therapeutic use.Accordingly, the preferred anti-CD47 antibodies described herein arecharacterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells; and

d. induces death of susceptible human tumor cells.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. induces death of susceptible human tumor cells; and

e. causes no agglutination of human red blood cells (hRBCs).

In yet another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. induces death of susceptible human tumor cells; and

e. causes reduced agglutination of human red blood cells (hRBCs).

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. specifically binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells

d. induces death of susceptible human tumor cells; and

e. has reduced hRBC binding.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

a. binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. causes no agglutination of human red blood cells (hRBCs); and

e. does not bind to hRBCs.

In another preferred embodiment described herein, the anti-CD47antibodies are characterized by:

-   -   a. specifically binds to human CD47,

b. blocks SIRPα binding to human CD47,

c. increases phagocytosis of human tumor cells,

d. causes no agglutination of human red blood cells (hRBCs); and

e. has reduced hRBC binding.

In another preferred embodiment described herein, the monoclonalantibody, or antigen binding fragment thereof also specifically binds tonon-human primate CD47, wherein non-human primate may include, but isnot limited to, cynomolgus monkey, green monkey, rhesus monkey andsquirrel monkey.

In yet another preferred embodiment described herein, the monoclonalantibody, or antigen binding fragment thereof binds human, non-humanprimate, mouse, rabbit, and rat CD47.

Described herein, are anti-CD47 mAbs with distinct functional profiles.These antibodies possess distinct combinations of properties selectedfrom the following: 1) exhibit cross-reactivity with one or more specieshomologs of CD47; 2) block the interaction between CD47 and its ligandSIRPα; 3) increase phagocytosis of human tumor cells, 4) induce death ofsusceptible human tumor cells; 5) do not induce cell death of humantumor cells; 6) have reduced binding to human red blood cells (hRBCs);7) have no detectable binding to hRBCs; 8) cause reduced agglutinationof hRBCs; 9) cause no detectable agglutination of hRBCs; 10) reverseTSP1 inhibition of the nitric oxide (NO) pathway and/or 11) do notreverse TSP1 inhibition of the NO pathway.

CD47 Antibodies

Many human cancers up-regulate cell surface expression of CD47 and thoseexpressing the highest levels of CD47 are appear to be the mostaggressive and the most lethal for patients. Increased CD47 expressionis thought to protect cancer cells from phagocytic clearance by sendinga “don't eat me” signal to macrophages via SIRPα, an inhibitory receptorthat prevents phagocytosis of CD47-bearing cells (Oldenborg et al.Science 288: 2051-2054, 2000; Jaiswal et al. (2009) Cell 138(2):271-851;Chao et al. (2010) Science Translational Medicine 2(63):63ra94). Thus,the increase of CD47 expression by many cancers provides them with acloak of “selfness” that slows their phagocytic clearance by macrophagesand dendritic cells.

Antibodies that block CD47 and prevent its binding to SIRPα have shownefficacy in human tumor in murine (xenograft) tumor models. Suchblocking anti-CD47 mAbs exhibiting this property increase thephagocytosis of cancer cells by macrophages, which can reduce tumorburden (Majeti et al. (2009) Cell 138 (2): 286-99; U.S. Pat. No.9,045,541; Willingham et al. (2012) Proc Natl Acad. Sci. USA109(17):6662-6667; Xiao et al. (2015) Cancer Letters 360:302-309; Chaoet al. (2012) Cell 142:699-713; Kim et al. (2012) Leukemia 26:2538-2545)and may ultimately lead to generation of an adaptive immune response tothe tumor (Tseng et al. (2013) PNAS 110 (27):11103-11108; Soto-Pantojaet al. (2014) Cancer Res. 74 (23): 6771-6783; Liu et al. (2015) Nat.Med. 21 (10): 1209-1215).

However, there are mechanisms by which anti-CD47 mAbs can attacktransformed cells that have not yet been exploited in the treatment ofcancer. Multiple groups have shown that particular anti-human CD47 mAbsinduce cell death of human tumor cells. Anti-CD47 mAb Ad22 induces celldeath of multiple human tumor cells lines (Pettersen et al. J. Immuno.166: 4931-4942, 2001; Lamy et al. J. Biol. Chem. 278: 23915-23921,2003). AD22 was shown to indice rapid mitochondrial dysfunction andrapid cell death with early phosphatidylserine exposure and a drop inmitochondrial membrane potential (Lamy et al. J. Biol. Chem. 278:23915-23921, 2003). Anti-CD47 mAb MABL-2 and fragments thereof inducecell death of human leukemia cell lines, but not normal cells in vitroand had an anti-tumor effect in in vivo xenograft models. (Uno et al.(2007) Oncol. Rep. 17 (5): 1189-94). Anti-human CD47 mAb 1F7 inducescell death of human T cell leukemias (Manna and Frazier (2003) J.Immunol. 170: 3544-53) and several breast cancers (Manna and Frazier(2004) Cancer Research 64 (3):1026-36). 1F7 kills CD47-bearing tumorcells without the action of complement or cell mediated killing by NKcells, T cells, or macrophages. Instead, anti-CD47 mAb 1F7 acts via anon-apoptotic mechanism that involves a direct CD47-dependent attack onmitochondria, discharging their membrane potential and destroying theATP-generating capacity of the cell leading to rapid cell death. It isnoteworthy that anti-CD47 mAb 1F7 does not kill resting leukocytes,which also express CD47, but only those cells that are “activated” bytransformation. Thus, normal circulating cells, many of which expressCD47, are spared while cancer cells are selectively killed by thetumor-toxic CD47 mAb (Manna and Frazier (2003) J. Immunol. 170:3544-53). This mechanism can be thought of as a proactive, selective anddirect attack on tumor cells in contrast to the passive mechanism ofcausing phagocytosis by simply blocking CD47/SIRPα binding. Importantly,mAb 1F7 also blocks binding of SIRPα to CD47 (Rebres et al et al. J.Cellular Physiol. 205: 182-193, 2005) and thus it can act via twomechanisms: (1) direct tumor toxicity, and (2) causing phagocytosis ofcancer cells. A single mAb that can accomplish both functions may besuperior to one that only blocks CD47/SIRPα binding.

Following periods of tissue ischemia, the initiation of blood flowcauses damage referred to as “ischemia-reperfusion injury” or IRI. IRIcontributes to poor outcomes in many surgical procedures where IRIoccurs due to the necessity to stop blood flow for a period of time, inmany forms/causes of trauma in which blood flow is interrupted and laterrestored by therapeutic intervention and in procedures required fororgan transplantation, cardio/pulmonary bypass procedures, reattachmentof severed body parts, reconstructive and cosmetic surgeries and othersituations involving stopping and restarting blood flow. Ischemia itselfcauses many physiological changes that, by themselves would eventuallylead to cell and tissue necrosis and death. Reperfusion poses its ownset of damaging events including generation of reactive oxygen species,thrombosis, inflammation and cytokine mediated damage. The pathways thatare limited by the TSP1-CD47 system are precisely those that would be ofmost benefit in combating the damage of IRI, including the NO pathway.Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosedherein, will provide more robust functioning of these endogenousprotective pathways. Anti-CD47 mAbs have been shown to reduce organdamage in rodent models of renal warm ishchemia (Rogers et al. J Am SocNephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion injury(Isenberg et al. Surgery. 144: 752-761, 2008), renal transplantation(Lin et al. Transplantation. 98: 394-401, 2014; Rogers et al. KidneyInterantional. 90: 334-347, 2016)) and liver transplantation, includingsteatotic livers (Xiao et al. Liver Transpl. 21: 468-477, 2015; Xiao etal. Transplantation. 100: 1480-1489, 2016). In addition, anti-CD47 mAbcaused significant reductions of right ventricular systolic pressure andright ventricular hypertrophy in the monocrotaline model of pulmonaryarterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693, 2012).Studies in skin flap models have shown that modulation of CD47,including with anti-CD47 mAbs, inhibits TSP1-mediated CD47 signaling.This results in inceased activity of the NO pathway, resulting inreduced IRI (Maxhimer et al. Plast Reconstr Surg. 124: 1880-1889, 2009;Isenberg et al. Arterioscler Throm Vasc Biol. 27: 2582-2588, 2007;Isenberg et al. Curr Drug Targets. 9: 833-841, 2008; Isenberg et al. AnnSurg. 247: 180-190, 2008) Anti-CD47 mAbs have also been shown to beefficacious in models of other cardiovascular diseases. In the mousetransverse aortic constriction model of pressure overload leftventricular heart failure, anti-CD47 mAb mitigated cardiac myocytehypertrophy, decreased left ventricular fibrosis, prevented an increasein left ventricular weight, decreased ventricular stiffness, andnormalized changes in the pressure volume loop profile (Sharifi-Sanjaniet al. J Am Heart Assoc., 2014). An anti-CD47 mAb amelioratedatherosclerosis in multiple mouse models (Kojima et al. Nature., 2016).

Cancer Indications

Presently disclosed are anti-CD47 mAbs and antigen binding fragmentsthereof effective as cancer therapeutics which can be administered topatients, preferably parenterally, with susceptible hematologic cancersand solid tumors including, but not limited to, leukemias, includingsystemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL),T cell—ALL, acute myeloid leukemia (AML), myelogenous leukemia, chroniclymphocytic leukemia (CLL), chronic myeloid leukemia (CML),myeloproliferative disorder/neoplasm, monocytic cell leukemia, andplasma cell leukemia; multiple myeloma (MM); Waldenstrom'sMacroglobulinemia; lymphomas, including histiocytic lymphoma and T celllymphoma, B cell lymphomas, including Hodgkin's lymphoma andnon-Hodgkin's lymphoma, such as low grade/follicular non-Hodgkin'slymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuselarge cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL; solid tumors, including ovariancancer, breast cancer, endometrial cancer, colon cancer (colorectalcancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer(non-small cell lung cancer, adenocarcinoma of the lung, squamous cellcarcinoma of the lung), bronchial cancer, bone cancer, prostate cancer,pancreatic cancer, gastric cancer, hepatocellular carcinoma (livercancer, hepatoma), gall bladder cancer, bile duct cancer, esophagealcancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma ofthe head and neck (head and neck cancer), testicular cancer, cancer ofthe endocrine gland, cancer of the adrenal gland, cancer of thepituitary gland, cancer of the skin, cancer of soft tissues, cancer ofblood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancerof meninges, cancer of oropharynx, cancer of hypopharynx, cancer ofcervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma,glioma, meningioma, gastrinoma, neuroblastoma, myelodysplastic syndrome,and sarcomas including, but not limited to, osteosarcoma, Ewing'ssarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma,angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, andchrondrosarcoma; and melanoma.

Treatment of Cancer

As is well known to those of ordinary skill in the art, combinationtherapies are often employed in cancer treatment as single-agenttherapies or procedures may not be sufficient to treat or cure thedisease or condition. Conventional cancer treatments often involvesurgery, radiation treatment, the administration of a combination ofcytotoxic drugs to achieve additive or synergistic effects, andcombinations of any or all of these approaches. Especially usefulchemotherapeutic and biologic therapy combinations employ drugs thatwork via different mechanisms of action, increasing cancer cell controlor killing, increasing the ability of the immune system to controlcancer cell growth, reducing the likelihood of drug resistance duringtherapy, and minimizing possible overlapping toxicities by permittingthe use of reduced doses of individual drugs.

Classes of conventional anti-tumor/anti-neoplastic agents useful in thecombination therapies encompassed by the present methods are disclosed,for example, in Goodman & Gilman's The Pharmacological Basis ofTherapeutics, Twelfth Edition (2010) L. L. Brunton, B. A. Chabner, andB. C. Knollmann Eds., Section VIII, “Chemotherapy of NeoplasticDiseases”, Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, and include,for example, alkylating agents, antimetabolites, natural products, avariety of miscellaneous agents, hormones and antagonists, targeteddrugs, monoclonal antibodies and other protein therapeutics.

In addition to the foregoing, the methods of the present disclosure arerelated to treatment of cancer indications and further comprisestreating the patient via surgery, radiation, and/or administering to apatient in need thereof an effective amount of a chemical small moleculeor biologic drug including, but not limited to, a peptide, polypeptide,protein, nucleic acid therapeutic, conventionally used or currentlybeing developed, to treat tumorous conditions. This includes antibodiesand antigen-binding fragments, other than those disclosed herein,cytokines, antisense oligonucleotides, siRNAs, and miRNAs.

The therapeutic methods disclosed and claimed herein include the use ofthe antibodies disclosed herein alone, and/or in combinations with oneanother, and/or with antigen-binding fragments thereof of the presentdisclosure that bind to CD47, and/or with competing antibodiesexhibiting appropriate biological/therapeutic activity, as well, forexample, all possible combinations of these antibody compounds toachieve the greatest treatment efficacy. In addition, the presenttherapeutic methods also encompass the use of these antibodies,antigen-binding fragments thereof, competing antibodies and combinationsthereof further in combination with: (1) any one or more anti-tumortherapeutic treatments selected from surgery, radiation, anti-tumor,anti-neoplastic agents, and combinations of any of these, or (2) any oneor more of anti-tumor biological agents, or (3) equivalents of any ofthe foregoing of (1) or (2) as would be apparent to one of ordinaryskill in the art, in appropriate combination(s) to achieve the desiredtherapeutic treatment effect for the particular indication.

Antibody and small molecule drugs that increase the immune response tocancer by modulating co-stimulatory or inhibitory interactions thatinfluence the T cell response to tumor antigens, including inhibitors ofimmune checkpoints and modulators of co-stimulatory molecules, are alsoof particular interest in the context of the combination therapeuticmethods encompassed herein and include, but are not limited to, otheranti-CD47 antibodies. Administration of therapeutic agents that bind tothe CD47 protein, for example, antibodies or small molecules that bindto CD47 and prevent interaction between CD47 and SIRPα, are administeredto a patient, causing the clearance of cancer cells via phagocytosis.The therapeutic agent that binds to the CD47 protein is combined with atherapeutic agent such as an antibody, a chemical small molecule orbiologic drug disclosed herein, directed against one or more additionalcellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2membrane glycoprotein, Cluster of Differentiation 200), CD154 (Clusterof Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation40 ligand), CD223 (Lymphocyte-activation gene 3, LAG3, Cluster ofDifferentiation 223), KIR (Killer-cell immunoglobulin-like receptors),GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein,activation-inducible TNFR family receptor, AITR, Tumor necrosis factorreceptor superfamily member 18), CD28 (Cluster of Differentiation 28),CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSFS, Tumornecrosis factor receptor superfamily member 5, p50), CD86 (B7-2, Clusterof Differentiation 86), CD160 (Cluster of Differentiation 160, BY55,NK1, NK28), CD258 (LIGHT, Cluster of Differentiation 258, Tumor necrosisfactor ligand superfamily member 14, TNFSF14, HVEML, HVEM ligand,herpesvirus entry mediator ligand, LTg), CD270 (HVEM, Tumor necrosisfactor receptor superfamily member 14, herpesvirus entry mediator,Cluster of Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOSligand, Inducible T-cell co-stimulator ligand, Cluster ofDifferentiation 275), CD276 (B7-H3, B7 homolog 3, Cluster ofDifferentiation 276), OX40L (OX40 Ligand), B7-H4 (B7 homolog 4, VTCN1,V-set domain-containing T-cell activation inhibitor 1), GITRL(Glucocorticoid-induced tumor necrosis factor receptor-ligand,glucocorticoid-induced TNFR-ligand), 4-1BBL (4-1BB ligand), CD3 (Clusterof Differentiation 3, T3D), CD25 (IL2Ra, Cluster of Differentiation 25,Interleukin-2 Receptor a chain, IL-2 Receptor a chain), CD48 (Cluster ofDifferentiation 48, B-lymphocyte activation marker, BLAST-1, signalinglymphocytic activation molecule 2, SLAMF2), CD66a (Ceacam-1,Carcinoembryonic antigen-related cell adhesion molecule 1, biliaryglycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80(B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation94), NKG2A (Natural killer group 2A, killer cell lectin-like receptorsubfamily D member 1, KLRD1), CD96 (Cluster of Differentiation 96,TActILE, T cell activation increased late expression), CD112 (PVRL2,nectin, Poliovirus receptor-related 2, herpesvirus entry mediator B,HVEB, nectin-2, Cluster of Differentiation 112), CD115 (CSF1R, Colonystimulating factor 1 receptor, macrophage colony-stimulating factorreceptor, M-CSFR, Cluster of Differentiation 115), CD205 (DEC-205, LY75,Lymphocyte antigen 75, Cluster of Differentiation 205), CD226 (DNAM1,Cluster of Differentiation 226, DNAX Accessory Molecule-1, PTA1,platelet and T cell activation antigen 1), CD244 (Cluster ofDifferentiation 244, Natural killer cell receptor 2B4), CD262 (DRS,TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB,ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor-4, TLR4, Cluster ofDifferentiation 284), CD288 (Toll-like Receptor-8, TLR8, Cluster ofDifferentiation 288), TNFSF15 (Tumor necrosis factor superfamily member15, Vascular endothelial growth inhibitor, VEGI, TL1A), TDO2 (Tryptophan2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type 1 Insulin-like GrowthFactor), GD2 (Disialoganglioside 2), TMIGD2 (Transmembrane andimmunoglobulin domain-containing protein 2), RGMB (RGM domain family,member B), VISTA (V-domain immunoglobulin-containing suppressor ofT-cell activation, B7-H5, B7 homolog 5), BTNL2 (Butyrophilin-likeprotein 2), Btn (Butyrophilin family), TIGIT (T cell Immunoreceptor withIg and ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-likelectins), Neurophilin, VEGFR (Vascular endothelial growth factorreceptor), ILT family (LIRs, immunoglobulin-like transcript family,leukocyte immunoglobulin-like receptors), NKG families (Natural killergroup families, C-type lectin transmembrane receptors), MICA (MHC classI polypeptide-related sequence A), TGFβ (Transforming growth factor (3),STING pathway (Stimulator of interferon gene pathway), Arginase(Arginine amidinase, canavanase, L-arginase, arginine transamidinase),EGFRvIII (Epidermal growth factor receptor variant III), and HHLA2(B7-H7, B7y, HERV-H LTR-associating protein 2, B7 homolog 7), inhibitorsof PD-1 (Programmed cell death protein 1, PD-1, CD279, Cluster ofDifferentiation 279), PD-L1 (B7-H1, B7 homolog 1, Programmeddeath-ligand 1, CD274, cluster of Differentiation 274), PD-L2 (B7-DC,Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster ofDifferentiation 273), CTLA-4 (Cytotoxic T-lymphocyte-associated protein4, CD152, Cluster of Differentiation 152), BTLA (B- and T-lymphocyteattenuator, CD272, Cluster of Differentiation 272), Indoleamine2,3-dioxygenase (IDO, IDO1), TIM3 (HAVCR2, Hepatitis A virus cellularreceptor 2, T cell immunoglobulin mucin-3, KIM-3, Kidney injury molecule3, TIMD-3, T cell immunoglobulin mucin-domain 3), A2A adenosine receptor(ADO receptor), CD39 (ectonucleoside triphosphate diphosphohydrolase-1,Cluster of Differentiation 39, ENTPD1), and CD73 (Ecto-5′-nucleotidase,5′-nucleotidase, 5′-NT, Cluster of Differentiation 73), CD27 (Cluster ofDifferentiation 27), ICOS (CD278, Cluster of Differentiation 278,Inducible T-cell Co-stimulator), CD137 (4-1BB, Cluster ofDifferentiation 137, tumor necrosis factor receptor superfamily member9, TNFRSF9), OX40 (CD134, Cluster of Differentiation 134), and TNFSF25(Tumor necrosis factor receptor superfamily member 25), includingantibodies, small molecules, and agonists, are also specificallycontemplated herein. Additional agents include IL-10 (Interleukin-10,human cytokine synthesis inhibitory factor, CSIF) and Galectins.

YERVOY® (ipilimumab; Bristol-Meyers Squibb) is an example of an approvedanti-CTLA-4 antibody.

KEYTRUDA® (pembrolizumab; Merck) and OPDIVO® (nivolumab; Bristol-MeyersSquibb Company) are examples of approved anti-PD-1 antibodies.

TECENTRIQ™ (atezolizumab; Roche) is an example of an approved anti-PD-L1antibody.

Ischemia-Reperfusion Injury (IRI)-Related, Autoimmune, Autoinflammatory,Inflammatory, Cardiovascular Conditions and Diseases

Administration of a CD47 mAb or antigen binding fragment thereofdisclosed herein can be used to treat a number of diseases andconditions in which IRI is a contributing feature, and to treat variousautoimmune, autoinflammatory, inflammatory and cardiovascular diseases.These include: organ transplantation in which a mAb or antigen bindingfragment thereof of the present invention is administered to the donorprior to organ harvest, to the harvested donor organ in the organpreservation solution, to the recipient patient, or to any combinationthereof; skin grafting; surgical resections or tissue reconstruction inwhich such mAb or fragment is administered either locally by injectionto the affected tissue or parenterally to the patient; reattachment ofbody parts; treatment of traumatic injury; pulmonary hypertension;pulmonary arterial hypertension; sickle cell disease (crisis);myocardial infarction; cerebrovascular disease; stroke;surgically-induced ischemia; acute kidney disease/kidney failure; anyother condition in which IRI occurs and contributes to the pathogenesisof disease; autoimmune and inflammatory diseases, including arthritis,rheumatoid arthritis, multiple sclerosis, psoriasis, psoriaticarthritis, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, lupus, systemic lupus erythematous, juvenile rheumatoidarthritis, juvenile idiopathic arthritis, Grave's disease, Hashimoto'sthyroiditis, Addison's disease, celiac disease, dermatomyositis,multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogrensyndrome, type I diabetes, vasculitis, uveitis and ankylosingspondylitis; autoinflammatory diseases, including familial Mediterraneanfever, neonatal onset multisystem inflammatory disease, tumor necrosisfactor (TNF) receptor-associated periodic syndrome, deficiency of theinterleukin-1 receptor antagonist, Behcet's disease; cardiovasculardiseases, including coronary heart disease, coronary artery disease,atherosclerosis, myocardial infarction, heart failure, and leftventricular heart failure.

Anti-CD47 mAbs and antigen binding fragments thereof of the presentinvention can also be used to increase tissue perfusion in a subject inneed of such treatment. Such subjects can be identified by diagnosticprocedures indicating a need for increased tissue perfusion. Inaddition, the need for increased tissue perfusion may arise because thesubject has had, is having, or will have, a surgery selected fromintegument surgery, soft tissue surgery, composite tissue surgery, skingraft surgery, resection of a solid organ, organ transplant surgery, orreattachment or an appendage or other body part.

Treatment of Ischemia-Reperfusion Injury (IRI)-Related Indications

The methods of the present disclosure, for example those related totreatment of IRI-related indications, can further comprise administeringto a patient in need thereof an effective amount of therapeutic agentthat binds to the CD47 protein and a nitric oxide donor, precursor, orboth; a nitric oxide generating topical agent; an agent that activatessoluble guanylyl cyclase; an agent that inhibits cyclic nucleotidephosphodiesterases; or any combination of any of the foregoing.

In these methods, the nitric oxide donor or precursor can be selectedfrom NO gas, isosorbide dinitrate, nitrite, nitroprus side,nitroglycerin, 3-Morpholinosydnonimine (SIN-1),S-nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO),S-nitrosothiols, Bidil®, and arginine.

The agent that activates soluble guanylyl cyclase can be a non-NO(nitric oxide)-based chemical activator of soluble guanylyl cyclase thatincreases cGMP levels in vascular cells. Such agents bind solubleguanylyl cyclase in a region other than the NO binding motif, andactivate the enzyme regardless of local NO or reactive oxygen stress(ROS). Non-limiting examples of chemical activators of soluble guanylylcyclase include organic nitrates (Artz et al. (2002) J. Biol. Chem.277:18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl.Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood84:4226-4233); BAY 41-2272 and BAY 41-8543 (Stasch et al. (2001 Nature410 (6825): 212-5), CMF-1571, and A-350619 (reviewed in Evgenov et al.(2006) Nat. Rev. Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Freyet al. (2008) Journal of Clinical Pharmacology 48 (12): 1400-10); BAY63-2521 (Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65).Additional soluble guanylyl cyclase activators are disclosed in Staschet al. (2011) Circulation 123:2263-2273; Derbyshire and Marletta (2012)Ann. Rev. Biochem. 81:533-559, and Nossaman et al. (2012) Critical CareResearch and Practice, Volume 2012, Article ID 290805, pages 1-12.

The agent that inhibits cyclic nucleotide phosphodiesterases can beselected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil.

Treatment of Autoimmune, Autoinflammatory, Inflammatory Diseases andCardiovascular Diseases

A therapeutic agent that binds to the CD47 protein for the treatment ofan autoimmune, autoinflammatory, inflammatory disease and/orcardiovascular disease can be combined with one or more therapeuticagent(s) such as an antibody, a chemical small molecule, or biologic ora medical or surgical procedure which include, but are not limited tothe following. For the treatment of autoimmune, autoinflammatory andinflammatory diseases, the combined therapeutic agents are:hydroxychloroquine, leflunomide, methotrexate, minocycline,sulfasalazine, abatacept, rituximab, tocilizumab, anti-TNF inhibitors orblockers (adalimumab, etanercept, infliximab, certolizumab pegol,golimumab), non-steroidal anti-inflammatory drugs, glucocorticoids,corticosteroids, intravenous immunoglobulin, anakinra, canakinumab,rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine,6-mercaptopurine, belimumab, beta interferons, glatiramer acetate,dimethyl fumarate, fingolimod, teriflunomide, natalizumab,5-aminosalicylic acid, mesalamine, cyclosporine, tacrolimus,pimecrolimus, vedolizumab, ustekinumab, secukinumab, ixekizumab,apremilast, budesonide and tofacitinib. For the treatment ofatherosclerosis, the combined therapeutic agents or procedures are:medical procedures and/or surgery, including percutaneous coronaryintervention (coronary angioplasty and stenting), coronary artery bypassgrafting, and carotid endarterectomy; therapeutic agents, includingangiotensin-converting enzyme (ACE) inhibitors (including ramipril,quinapril, captopril, and enalapril), calcium channel blockers(including amiodipine, nifedipine, verapamil, felodipine and diltiazem),angiotensin-receptor blockers (including eposartan, olmesarten,azilsartan, valsartan, telmisartan, losartan, candesartan, andirbesartan), the combination of ezetimibe and simvastatin, PCSK9inhibitors (including alirocumab and evolocumab), anacetrapib, andHMG-CoA inhibitors (including atorvastatin, pravastatin, simvastatin,rosuvastatin, pitavastatin, lovastatin and fluvastatin). For thetreatment of heart failure, the combined therapeutic agents are: ACEinhibitors, angiotensin receptor blockers, angiotensin receptorneprilsyn inhibitors (including the combination of sacubitril andvalsartan), diuretics, digoxin, inotropes, beta blockers and aldosteroneantagonists. For the treatment of pumonary hypertension the combinedtherapeutic agents are: sildenafil, tadalafil, ambrisentan, bosentan,macitentan, riociguat, treprostinil, epoprostenol, iloprost, andselexipag.

As disclosed herein, the anti-CD47 mAb is administered before, at thesame time or after the combined therapeutic agents or medical orsurgical procedures.

Another useful class of compounds for the combination therapiescontemplated herein includes modulators of SIRPα/CD47 binding such asantibodies to SIRPα, as well as soluble protein fragments of thisligand, or CD47 itself, inhibiting binding of, or interfering withbinding of, SIRPα to CD47. It should be noted that the therapeuticmethods encompassed herein include the use of the antibodies disclosedherein alone, in combination with one another, and/or withantigen-binding fragments thereof as well, for example, all possiblecombinations of these antibody compounds.

The examples illustrate various embodiments of the present disclosure,but should not be considered as limiting the disclosure to only theseparticularly disclosed embodiments.

Diagnostics for CD47 Expression

Diagnostics (including complementary and companion) have been an area offocus in the field of oncology. A number of diagnostic assays have beendeveloped for targeted therapeutics such as Herceptin (Genentech),Tarceva (OSI Pharmaceuticals/Genentech), Iressa (Astra Zeneca), andErbitux (Imclone/Bristol Myers Squibb). The anti-CD47 mAbs antibodies ofthe disclosure are particularly well-suited to use in diagnosticapplications. Accordingly, the disclosure provides a method to measureCD47 expression in tumor and/or immune cells, using an anti-CD47 mAb ofthe disclosure.

The anti-CD47 mAbs of the disclosure may be used in a diagnostic assayand/or in vitro method to measure CD47 expression in tumor and/or immunecells present in a patient's tumor sample. In particular, the anti-CD47mAbs of the disclosure may bind CD47 on approximately 1% or more oftumor and/or immune cells present in a patient's sample as compared to areference level. In another embodiment, the anti-CD47 mAbs may bind CD47on approximately 5% or more of tumor and/or immune cells in a patient'ssample as compared to a reference level, for example, or binding atleast 10%, or at least 20%, or at least 30%, or at least about 40%, orat least about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or between 10-100% as comparedto a reference level. In yet another embodiment, the anti-CD47 mAbs maybind CD47 on tumor and/or immune cells in a patient's sample to at leastabout a 2-fold increase as compared to a reference level, or at leastabout 3-fold, or at least about a 4-fold, or at least about a 5-fold orat least about a 10-fold increase, or between 2-fold and 10-fold orgreater as compared to a reference level. As described herein, themeasurement of CD47 expression in a patient's sample provides biologicaland/or clinical information that enables decision making about thedevelopment and use of a potential drug therapy, notably the use ofanti-CD47 antibodies for treating solid and hematological cancers,autoimmune disease, inflammatory disease, atherosclerosis, heartfailure, in which the CD47 receptor plays a role.

In one embodiment, the in vitro method comprises, obtaining a patientsample, contacting the patient sample with a monoclonal antibody, orantigen-binding fragment thereof, which specifically binds to an epitopewithin the sequence of SEQ ID NO:66, and assaying for binding of theantibody to the patient sample, wherein binding of the antibody to thepatient sample is diagnostic of CD47 expression in a patient sample.

In another embodiment, the preferred CD47 antibodies, or antigen bindingfragments thereof, for the in vitro method, are those comprising acombination of a heavy chain (HC) and a light chain (LC), listed fromthe combination of:

-   -   (i) a heavy chain comprising the amino acid sequence of SEQ ID        NO:103 and a light chain comprising the amino acid sequence SEQ        ID NO:102;    -   (ii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:105 and a light chain comprising the amino acid sequence SEQ        ID NO:104;    -   wherein the V_(H) amino acid sequence is at least 90%, 95%, 97%,        98% or 99% identical thereto and the a V_(L) amino acid sequence        is at least 90%, 95%, 97%, 98% or 99% identical thereto.

Accordingly, a diagnostic assay in accordance with the disclosure maycomprise contacting tumor and/or immune cells in a patient's sample withan anti-CD47 mAb, or an antigen binding fragment thereof, and assayingfor binding of the anti-CD47 mAb to a patient's tumor sample, whereinbinding of the anti-CD47 mAb to the patient sample is diagnostic of CD47expression. Preferably, the patient's sample is a sample containingtumor cells. In this case, binding of the anti-CD47 mAb of thedisclosure, or antigen binding fragment thereof, to the tumor cells maybe assessed for CD47 expression. The levels of CD47 expression by tumorcells and/or immune cells of a patient's tumor sample may be predictiveof clinical outcome in a patient.

Increased binding of anti-CD47 mAbs binding to cells in a patient'ssample is associated with increased CD47 expression. In one embodiment,the anti-CD47 mAbs of the disclosure may bind to approximately 5% ormore of tumor cells in a patient's sample and this may indicate that thepatient would benefit from rapid intervention to a solid andhematological cancer. A diagnostic assay of this sort may be used todetermine suitable therapeutic regimes for solid and hematologicalcancers with relatively high binding of anti-CD47 mAbs of thedisclosure, i.e., increased CD47 expression.

It will be appreciated that the diagnostic assay disclosed herein has anumber of advantages. The most important of these advantages is that thediagnostic assay of the disclosure may allow the user a greater deal ofconfidence in the CD47 expression in tumor and/or immune cells. Theincreased sensitivity of the diagnostic assay of the disclosure allowsdetection of CD47 in a patient's sample at lower levels than haspreviously been the case.

The anti-CD47 mAbs of the disclosure may be used as a diagnostic assayin relation to many forms of cancer. Particular forms of cancer that mayadvantageously be investigated for CD47 expression include susceptiblehematologic cancers and solid tumors including, but not limited to,leukemias, lymphomas, and solid tumors.

The diagnostic assays of the disclosure may utilize any suitable meansfor detecting binding of an anti-CD47 mAb to measure CD47 expression.Suitable methods may be selected with reference to the nature of anyreporter moiety used to label the anti-CD47 mAbs of the disclosure.Suitable techniques include, but are by no means limited to, flowcytometry, and enzyme linked immunosorbent assays (ELISA) and assaysutilizing nanoparticles. It is particularly preferred that a diagnosticassay of the invention be one involving immunohistochemistry in which atumor sample is exposed to an anti-CD47 mAb of the disclosure, and thelevel of cell labelling is assessed by immunohistochemistry.

EXAMPLES Example 1 Amino Acid Sequences Light Chain CDRs

LCDR1 LCDR2 LCDR3 Vx4-LCDR1 Vx4-LCDR2 Vx4-LCDR3 RSRQSIVHTNGNTYLG KVSNRFSFQGSHVPYT (SEQ ID NO: 11) (SEQ ID NO: 15) (SEQ ID NO: 18) Vx8-LCDR1Vx8-LCDR2 Vx8-LCDR3 RASQDISNYLN YTSRLYS QQGNTLPWT (SEQ ID NO: 12)(SEQ ID NO: 16) (SEQ ID NO: 19) Vx8-LCDR1 RASQSISNYLN (SEQ ID NO: 13)Vx9-LCDR1 Vx9-LCDR2 Vx9-LCDR3 RSSQNIVQSNGNTYLE KVFHRFS FQGSHVPWT(SEQ ID NO: 14) (SEQ ID NO: 17) (SEQ ID NO: 20)

Heavy Chain CDRs

HCDR1 HCDR2 HCDR3 Vx4-HCDR1 Vx4-HCDR2 Vx4-HCDR3 GYTFTNYVIHYIYPYNDGILYNEKFKG GGYYVPDY (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 7)Vx4-HCDR3 GGYYVYDY (SEQ ID NO: 8) Vx8-HCDR1 Vx8-HCDR2 Vx8-HCDR3GYSFTNYYIH YIDPLNGDTTYNQKFKG GGKRAMDY (SEQ ID NO: 2) (SEQ ID NO: 5)(SEQ ID NO: 9) Vx9-HCDR1 Vx9-HCDR2 Vx9-HCDR3 GYTFTNYWIHYTDPRTDYTEYNQKFKD GGRVGLGY (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 10)

Murine Light Chain Variable Domains

>Vx4murL01 (SEQ ID NO: 41)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK. >Vx4murL02 (SEQ ID NO: 42)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIK. >Vx8murL03 (SEQ ID NO: 46)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPW TFGGGTKLEIK. >Vx9murL04(SEQ ID NO: 50) DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQG SHVPWTFGGGTKVEIK

Murine Heavy Chain Variable Domains

>Vx4murH01 (SEQ ID NO: 21)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSS. >Vx4mur-H02 (SEQ ID NO: 22)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTLVTVSS. >Vx8murH03 (SEQ ID NO: 28)EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSS. >Vx9murH04 (SEQ ID NO: 35)QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYC AGGGRVGLGYWGHGSSVTVSS

Human Light Chain Variable Domains

>Vx4humL01 (SEQ ID NO: 43)DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIK >Vx4humL02 (SEQ ID NO: 44)DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK >Vx4humL03 (SEQ ID NO: 45)DIVMTQSPDSLAVSLGERATINCRSRQSIVHTNGNTYLGWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIK >Vx8humL04 (SEQ ID NO: 47)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPW TFGQGTKVEIK. >Vx8humL05(SEQ ID NO: 48) DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPW TFGQGTKVEIK. >Vx8humL06(SEQ ID NO: 49) DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPW TFGQGTKLEIK >Vx9humL07(SEQ ID NO: 51) DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK. >Vx9humL08 (SEQ ID NO: 52)DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQG SHVPYTFGQGTKLEIK.

Human Heavy Chain Variable Domains

>Vx4humH01 (SEQ ID NO: 23)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSS. >Vx4humH02 (SEQ ID NO: 24)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSS. >Vx4humH03 (SEQ ID NO: 25)EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYVIHWVQQAPGKGLEWMGYIYPYNDGILYNEKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYYVPDYWGQGTTVTVSS >Vx4humH04 (SEQ ID NO: 26)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSS >Vx4humH05 (SEQ ID NO: 27)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSS >Vx8humH06 (SEQ ID NO: 29)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH07 (SEQ ID NO: 30)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH08 (SEQ ID NO: 31)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH09 (SEQ ID NO: 32)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH10 (SEQ ID NO: 33)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSS. >Vx8humH11 (SEQ ID NO: 34)QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSS >Vx9humH12 (SEQ ID NO: 36)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH13 (SEQ ID NO: 37)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH14 (SEQ ID NO: 38)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH15 (SEQ ID NO: 39)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH16 (SEQ ID NO: 40)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC ARGGRVGLGYWGQGTLVTVSS.

Human IgG-Fc

>Human Fc IgG1 (SEQ ID NO: 53)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc IgG1-N297Q(SEQ ID NO: 54) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG2(SEQ ID NO: 56) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG3 (SEQ ID NO: 57)ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK >Human Fc-IgG4 (SEQ ID NO: 58)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4 S228P(SEQ ID NO: 59) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4 PE(SEQ ID NO: 60) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >Human Fc-IgG4 PE′(SEQ ID NO: 101) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG >Human kappa LC (SEQ ID NO: 61)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Rat Fc-IgG2c (SEQ ID NO: 62)ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK. >Rat kappa LC (SEQ ID NO: 63)RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSS PVVKSFNRNEC.

Rabbit IgG-Fc

>Rabbit IgG (SEQ ID NO: 64)GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINTNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINTGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK. >Rabbit kappa LC (SEQ ID NO: 65)RDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVV QSFNRGDC. >CD47(SEQ ID NO: 66) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVY MKFVE.

Chimera and Human Light Chains

>Vx4murL01 Full length (SEQ ID NO: 67)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4murL01 Full length (SEQ ID NO: 68)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4humL01 Full length LC (SEQ ID NO: 69)DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL03 Full length LC (SEQ ID NO: 70)DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9humL02 Full length LC (SEQ ID NO: 71)DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL02 Full length LC (SEQ ID NO: 72)DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4humL02 Full length LC (SEQ ID NO: 73)DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9humL07 Full length LC (SEQ ID NO: 74)DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL01 Full length LC (SEQ ID NO: 75)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8murL03 Full length LC (SEQ ID NO: 76)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9mur L04 Full length LC (SEQ ID NO: 77)DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Chimera and Human Heavy Chains

>Vx4murH01 Full length HC (SEQ ID NO: 78)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH01 Full length HC (SEQ ID NO: 79)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH11 Full length HC (SEQ ID NO: 80)QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH12 Full length HC (SEQ ID NO: 81)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH14 Full length HC (SEQ ID NO: 82)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH15 Full length HC (SEQ ID NO: 83)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx4humH02 Full length HC (SEQ ID NO: 84)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH13 Full length HC (SEQ ID NO: 85)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH10 Full length HC (SEQ ID NO: 86)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH04 Full length HC (SEQ ID NO: 87)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH05 Full length HC (SEQ ID NO: 88)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH16 Full length HC (SEQ ID NO: 89)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full length HC (SEQ ID NO: 90)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH07 Full length HC (SEQ ID NO: 91)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH08 Full length HC (SEQ ID NO: 92)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH09 Full length HC (SEQ ID NO: 93)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH06 Full length HC (SEQ ID NO: 94)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8mur-H03 Full length HC (SEQ ID NO: 95)EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9mur-H04 Full length HC (SEQ ID NO: 96)QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGSSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full length HC (SEQ ID NO: 97)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH07 Full length HC (SEQ ID NO: 98)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH08 Full length HC (SEQ ID NO: 99)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH09 Full length HC (SEQ ID NO: 100)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx4mur-ratL01 Full length LC (SEQ ID NO: 102)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVKSFNRNEC. >Vx4mur-ratH01 Full length HC(SEQ ID NO: 103) EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSSARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK. >Vx4mur-rabL01 Full length LC (SEQ ID NO: 104)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC. >Vx4mur-rabH01 Full length HC (SEQ ID NO: 105)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINTNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQ KSISRSPGK.

Example 2 Production of CD47 Antibodies

Chimeric antibodies disclosed herein comprise a mouse heavy chainvariable domain and a light chain variable domain combined with a humankappa or human Fc IgG1, IgG1-N297Q, IgG2, IgG4, IgG4 S228P, and IgG4 PEconstant domains, respectively. These were designed to incorporate asecretion signal and cloned into a mammalian expression system, andtransfected into CHO cells to generate chimeric (murine-human)antibodies. The chimeric variants were expressed as full length IgGmolecules, secreted into the medium, and purified using protein A.

As such, the humanized antibodies disclosed herein comprise frameworksderived from the human genome. The collection covers the diversity foundin the human germ line sequences, yielding functionally expressedantibodies in vivo. The complementarity determining regions (CDRs) inthe light and heavy chain variable regions of the murine and chimeric(murine-human) are described herein and were determined by followingcommonly accepted rules disclosed in “Protein Sequence and StructureAnalysis of Antibody Variable Domains”, In: Antibody Engineering LabManual, eds. S. Duebel and R. Kontermann, Springer-Verlag, Heidelberg(2001)). The human light chain variable domains were then designed. Thehumanized variable domains were then combined with a secretion signaland human kappa and human Fc IgG1, IgG1-N297Q, IgG2, IgG3, IgG4 S228Pand IgG4 PE constant domains, cloned into a mammalian expression system,and transfected into CHO cells to generate humanized mAbs. The humanizedvariants were expressed as full length IgG molecules, secreted into themedium and purified using protein A.

A non-glycosylated version (IgG1-N297Q) was created by site directedmutagenesis of heavy chain position 297 to change the asparagine toglutamine (Human Fc IgG1-N297Q, SEQ ID NO:54). An IgG4 variant wascreated by site-directed mutagenesis at position 228 to change theserine to proline thereby preventing in vivo Fab arm exchange. An IgG4double mutant was created by site-directed mutagenesis at positions 228(serine to proline) and 235 (leucine to glutamate) to prevent Fab armexchange and to further reduce Fc effector function. IgG2, IgG3, IgG4S228P, and IgG4 PE isotypes were constructed by cloning the heavy chainvariable domain in frame with the human IgG2, IgG3, IgG4 S228P, andIgG4PE constant domains (Human Fc-IgG2, SEQ ID NO:56 Human Fc-IgG3, SEQID NO:57; Human Fc-IgG4 S228P, SEQ ID NO:59; and Human Fc-IgG4 PE, SEQID NO:60).

Example 3 Binding of CD47 Monoclonal Antibodies (mAbs)

The binding of chimeric (murine-human) and humanized antibodies of thepresent disclosure was determined by ELISA using OV10 cells transfectedwith human CD47 (OV10 hCD47) or using freshly isolated human red bloodcells (hRBCs), which display CD47 on their surface (Kamel et al. (2010)Blood. Transfus. 8(4):260-266).

Binding activities of VLX4, VLX8, and VLX9 chimeric (murine-human) andhumanized mAbs were determined using a cell-based ELISA assay with humanOV10 hCD47 cells expressing cell surface human CD47. OV10 hCD47 cellswere grown in IMDM medium containing 10% heat inactivated fetal bovineserum (BioWest; S01520). One day before assay, 3×10⁴ cells were platedin 96 well cell bind plates (Corning #3300, VWR #66025-626) and were95-100% confluent at the time of assay. Cells were washed and variousconcentrations of purified antibodies added in IMDM 37° C. for 1 hr in95% O₂/5% CO₂. Cells were then washed with media and incubated for anadditional hour at 37° C. with HRP labeled secondary anti-human antibody(Promega) diluted 1/2500 in media. Cells were washed three times withPBS, and the peroxidase substrate 3,3′, 5,5′-tetramethylbenzidine isadded (Sigma; Catalog #T4444). Reactions were terminated by the additionof HCl to 0.7N, and absorbance at 450 nM is determined using a Tecanmodel Infinite M200 plate reader. The apparent binding affinities ofthese clones to human OV10 hCD47 cells was determined by non-linear fit(Prism GraphPad software).

Binding activities of chimeric (murine-human) and humanized VLX4, VLX8,and VLX9 mAbs to human CD47 on hRBCs were also determined using flowcytometry. hRBCs were incubated for 60 min on at 37° C. with variousconcentrations of the chimeric or humanized antibodies in a solution ofphosphate buffered saline, pH 7.2, 2.5 mM EDTA (PBS+E). Cells were thenwashed with cold PBS+E, and incubated for an additional hour on ice withFITC labeled donkey anti-human antibody (Jackson Immuno Research Labs,West Grove, Pa.; Catalogue #709-096-149) in PBS+E. Cells were washedwith PBS+E, antibody binding analyzed using a C6 Accuri Flow Cytometer(Becton Dickinson) and apparent binding affinities determined bynon-linear fit (Prism GraphPad software) of the median fluorescenceintensities at the various antibody concentrations.

All of the VLX4 chimeric (murine-human) mAbs bound to human OV10 hCD47tumor cells with apparent affinities in the picomolar (pM) range (Table1).

Similarly, the humanized VLX4 mAbs bound to human OV10 hCD47 tumor cellsin a concentration-dependent manner (FIG. 1A and FIG. 1B) with apparentaffinities ranging from the picomolar to low nanomolar range (Table 2).

All of the chimeric VLX4 mAbs bound to human RBCs with apparent K_(d)values in the picomolar range and these were similar to the K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1).

The humanized VLX4 mAbs VLX4hum_01 IgG1 N297Q, VLX4hum_02 IgG1 N297Q,VLX4hum_01 IgG4 PE, VLX4hum_02 IgG4 PE, VLX4hum_12 IgG4 PE, andVLX4hum_13 IgG4 bound to human RBCs with K_(d) values similar to thevalues obtained for OV10 hCD47 tumor cells whereas VLX4hum_06 IgG4 PEand VLX4hum_07 IgG4 PE exhibited reduced binding to hRBCs (FIG. 2A, FIG.2B, and Table 2). This differential binding of the humanized mAbs totumor cells and RBCs was unexpected as the VLX4 IgG4PE chimeric mAbbound with similar apparent K_(d) values to both tumor and RBC CD47(Table 1).

As shown in Table 1, all of the VLX8 chimeric (murine-human) mAbs boundto human OV10 hCD47 tumor cells in a concentration-dependent manner withapparent affinities in the picomolar (pM) range.

Similarly, the humanized VLX8 mAbs bound to human OV10 hCD47 tumor cellsin a concentration-dependent manner (FIG. 3A and FIG. 3B) with apparentaffinities in the picomolar range (Table 2).

All of the VLX8 chimeric mAbs bound to hRBCs with apparent K_(d) valuesin the picomolar range and these were similar to the K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1).

The VLX8 humanized mAbs VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE,VLX8hum_03 IgG4 PE, VLX8hum_04 IgG4 PE, VLX8hum_05 IgG4 PE, andVLX8hum_06 IgG4 PE, VLX8hum_07 IgG4 PE, VLX8hum_08 IgG4 PE, VLX8hum_09IgG4 PE, VLX8hum_11 IgG4 PE, VLX8hum_06 IgG2, VLX8hum_07 IgG2,VLX8hum_08 and VLX8hum_09 IgG2 IgG2 bound to human RBCs with K_(d)values similar to the values obtained for OV10 hCD47 tumor cells whereasVLX8hum_10 IgG4 PE exhibited reduced, but measurable binding to hRBCs(FIG. 4A, FIG. 4B, and Table 2). This differential binding of thehumanized mAbs to tumor cells and RBCs was unexpected as the VLX8 IgG4PEchimeric mAb bound with similar apparent K_(d) values to both tumor andRBC CD47 (Table 1).

Table 1 shows the apparent binding affinities of VLX9 murine-humanchimeric mAbs to human OV10 hCD47 cells and to human RBCs. All of thechimeric mAbs bound to OV10 hCD47 tumor cells with apparent K_(d) valuesin the picomolar range. Similarly, the humanized VLX9 mAbs bound tohuman OV10 hCD47 tumor cells in a concentration-dependent manner (FIG.5A and FIG. 5B) with apparent affinities in the picomolar to nanomolarrange (Table 2).

All of the VLX9 chimeric mAbs bound to hRBCs with apparent K_(d) valuesin the picomolar range and these were similar to the K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1). In contrast tothe chimeric mAbs, the VLX9 humanized mAbs VLX9hum_01 IgG2, VLX9hum_(—)02 IgG2 and VLX9hum_07 IgG2 exhibited reduced but measurable binding tohuman RBCs (FIG. 6, Table 2). Humanized mAbs VLX9hum_03, 04, 05, 06, 08,09 and 10 IgG2 exhibited no measurable binding to RBCs (Table 2). Thisdifferential binding of the humanized mAbs to tumor cells and RBCs wasunexpected as the VLX9 IgG2 chimeric mAbs all bound with similarapparent K_(d) values to both tumor and RBC CD47 (Table 1).

TABLE 1 Binding of VLX4, VLX8, and VLX9 Chimeric (xi) mAbs to OV10 hCD47Cells and Human Red Blood Cells (hRBCs). Kd (pM) V_(H) V_(L) OV10 (SEQ(SEQ hCD47 ID ID Cell-based Kd (pM) HA NO:) NO:) ELISA hRBC hRBC VLX4IgG1 (xi) 21 41 315 104 Yes VLX4 IgG1 N297Q (xi) 21 41 258 92 Yes VLX4IgG2 (xi) 21 41 431 184 Yes VLX4 IgG4 S228P (xi) 21 41 214 99 No VLX4IgG4 PE (xi) 21 41 225 303 No VLX8 IgG1 N297Q (xi) 28 46 42 91 Yes VLX8IgG4 PE (xi) 28 46 56 77 Yes VLX9 IgG1 (xi) 35 50 280 381 Yes VLX9 IgG1N297Q (xi) 35 50 275 190 Yes VLX9 IgG2 (xi) 35 50 880 742 Yes VLX9 IgG4PE (xi) 35 50 293 126 Yes

TABLE 2 Binding of VLX4, VLX8, and VLX9 Humanized mAbs to Human OV10hCD47 and Human Red Blood Cells (hRBCs). Kd (pM) OV10 hCD47 Cell-basedKd (pM) HA ELISA hRBC hRBC VLX4hum_01 IgG1 73  23 Yes VLX4hum_02 IgG1 80 70 Yes VLX4hum_01 IgG4 PE 82  63 No VLX4hum_02 IgG4 PE 95  75 R***VLX4hum_06 IgG4 PE 196 >66,000**  Yes VLX4hum_07 IgG4 PE 209 >66,000** Yes VLX4hum_12 IgG4 PE 56 263 Yes VLX4hum_13 IgG4 PE 62 340 YesVLX8hum_01 IgG4 PE 54 209 No VLX8hum_02 IgG4 PE 50 221 No VLX8hum_03IgG4 PE 67 183 No VLX8hum_04 IgG4 PE 49 119 No VLX8hum_05 IgG4 PE 68 264No VLX8hum_06 IgG4 PE 61 274 Yes VLX8hum_07 IgG4 PE 24 241 YesVLX8hum_08 IgG4 PE 97 217 Yes VLX8hum_09 IgG4 PE 82 336 Yes VLX8hum_10IgG4 PE 183 >33,000**  Yes VLX8hum_11 IgG4 PE 90  18 No VLX8hum_06 IgG2403 246 Yes VLX8hum_07 IgG2 460 671 Yes VLX8hum_08 IgG2 464 820 YesVLX8hum_09 IgG2 680 1739  Yes VLX9hum_01 IgG2 162  1653** N VLX9hum_02IgG2 227  4103** N VLX9hum_03 IgG2 606 *NB N VLX9hum_04 IgG2 823 *NB NVLX9hum_05 IgG2 6372 *NB N VLX9hum_06 IgG2 547 *NB N VLX9hum_07 IgG2 341>66,000**  ***R VLX9hum_08 IgG2 688 *NB N VLX9hum_09 IgG2 8340 *NB NVLX9hum_10 IgG2 12232 *NB N *NB—No binding detected at mAb concentrationup to 100 μg/mL. **Reduced RBC binding. ***R—Reduced hemagglutination.

Cross-species binding of humanized VLX4, VLX8, and VLX9 mAbs wasdetermined using flow cytometry. Mouse, rat, rabbit or cynomolgus monkeyRBCs were incubated for 60 min on at 37° C. with various concentrationsof the humanized antibodies in a solution of phosphate buffered saline,pH 7.2, 2.5 mM EDTA (PBS+E). Cells were then washed with cold PBS+E, andincubated for an additional hr on ice with FITC labeled donkeyanti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.;Catalogue #709-096-149) in PBS+E. Cells were washed with PBS+E, andantibody binding analyzed using a C6 Accuri Flow Cytometer (BectonDickinson).

Table 3 shows the apparent binding affinities of the humanized VLX4 andVLX8 mAbs to RBCs from mouse, rat, and cynomolgus monkey determined bynon-linear fit (Prism GraphPad software) of the median fluorescenceintensities at various antibody concentrations. This data demonstratesthat humanized VLX4 and VLX8 mAbs bind to mouse, rat, rabbit (data notshown) and cynomolgus monkey RBCs with apparent Kd values in thepicomolar to nanomolar range (Table 4).

TABLE 3 Binding of VLX4 and VLX8 Humanized mAbs to mouse and rat RBCs.Kd (pM) Kd (pM) Kd (pM) Cynomolgus Mouse Rat Monkey RBC RBC RBCVLX4hum_01 IgG4 PE 16166 29917 23 VLX4hum_07 IgG4 PE 21340 17610 4313VLX8hum_11 IgG4PE 2473 10921 76

Example 4 CD47 Antibodies Block CD47/SIRPα Binding

To assess the effect of humanized CD47 mAbs on binding of CD47 to SIRPαin vitro the following method is employed using the binding offluorescently-labelled SIRPα-Fc fusion protein to CD47 expressing JurkatT cells.

SIRPα-Fc fusion protein (R&D Systems, cat #4546-SA) was labelled usingan Alexa Fluor® antibody labelling kit (Invitrogen Cat No. A20186)according to the manufacturers specifications. 1.5×10⁶ Jurkat T cellswere incubated with humanized mAbs (5 μg/ml), a human control antibodyin RPMI containing 10% media or media alone for 30 min at 37° C. Anequal volume of fluorescently labeled SIRPα-Fc fusion protein was addedand incubated for an additional 30 min at 37° C. Cells were washed oncewith PBS and the amount of labelled SIRPα-Fc bound to the Jurkat T cellsanalyzed by flow cytometry.

As shown in FIG. 7, the humanized VLX4, VLX8 and VLX9 mAbs, blocked theinteraction of CD47 expressed on the Jurkat T cells with SIPRα, whilethe human control antibody (which does not bind to CD47) or media alone,did not block the CD47/SIRPα interaction.

Example 5 CD47 Antibodies Increase Phagocytosis

To assess the effect of chimeric (murine-human) and humanized VLX4,VLX8, and VLX9 CD47 mAbs on phagocytosis of tumor cells by macrophagesin vitro the following method is employed using flow cytometry(Willingham et al. (2012) Proc Natl Acad Sci USA 109(17):6662-7 andTseng et al. (2013) Proc Natl Acad Sci USA 110(27):11103-8).

Human derived macrophages were derived from leukapheresis of healthyhuman peripheral blood and incubated in AIM-V media (Life Technologies)for 7-10 days. For the in vitro phagocytosis assay, macrophages werere-plated at a concentration of 1×10⁴ cells per well in 100 ul of AIM-Vmedia in a 96-well plate and allowed to adhere for 24 hrs. Once theeffector macrophages adhered to the culture dish, the target humancancer cells (Jurkat) were labeled with 1 μM 5(6)-Carboxyfluoresceindiacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to themacrophage cultures at a concentration of 5×10⁴ cells in 1 ml of AIM-Vmedia (5:1 target to effector ratio). VLX4, VLX8, and VLX9 CD47 mAbs (1μg/ml) were added immediately upon mixture of target and effector cellsand allowed to incubate at 37° C. for 2-3 hours. After 2-3 hrs, allnon-phagocytosed cells were removed and the remaining cells washed threetimes with phosphate buffered saline (PBS; Sigma Aldrich). Cells werethen trypsinized, collected into microcentrifuge tubes, and incubated in100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD Biosciences)for 30 minutes, washed once, and analyzed by flow cytometry (Accuri C6;BD Biosciences) for the percentage of CD14+ cells that were also CFSEindicating complete phagocytosis.

As shown in FIG. 8, the VLX4 chimeric (murine-human) mAbs VLX4 IgG1,VLX4 IgG1 N297Q, VLX4 IgG4 PE, and VLX4 IgG4 S228P increasedphagocytosis of Jurkat cells by human macrophages by blocking theCD47/SIRPα interaction and this enhanced phagocytosis is independent ofFc function.

Similarly, as shown in FIG. 9A and FIG. 9B, VLX4hum_01 IgG1, VLX4hum_01IgG4 PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, andVLX4hum_13 IgG4 PE increased phagocytosis of Jurkat cells by humanmacrophages by blocking the CD47/SIRPα interaction.

As shown in FIG. 10A, the VLX8 chimeric (murine-human) mAbs VLX8 IgG1N297Q and VLX8 IgG4 PE increase phagocytosis of Jurkat T cells by humanmacrophages via blocking the CD47/SIRPα interaction and this enhancedphagocytosis is independent of Fc function.

Similarly, as shown in FIG. 10B, VLX8hum_01 IgG4 PE, VLX8hum_03 IgG4 PE,VLX8hum_07 IgG4 PE, VLX8hum_08 IgG4 PE, and VLX8hum_09 IgG4 PE increasedphagocytosis of Jurkat cells by human macrophage by blocking theCD47/SIRPα interaction and this enhanced phagocytosis is independent ofFc function

As shown in FIG. 11A, the VLX9 IgG1N297Q, VLX9 IgG2 and VLX9 IgG4 PEchimeric mAbs all increased phagocytosis of Jurkat T cells by humanmacrophages by blocking the CD47/SIRPα interaction and this enhancedphagocytosis is independent of Fc effector function. Similarly as shownin FIG. 11B, all of the humanized VLX9 IgG2 mAbs increased phagocytosisof Jurkat T cells.

Example 6 Induction of Cell Death by Soluble CD47 Antibodies

Some soluble CD47 antibodies have been shown to induce selective celldeath of tumor cells. This additional property of selective toxicity tocancer cells is expected to have advantages compared to mAbs that onlyblock SIRPα binding to CD47.

Induction of cell death by soluble anti-CD47 mAbs is measured in vitro(Manna et al. (2003) J. Immunol. 107 (7): 3544-53). For the in vitrocell death assay, 1×10⁵ transformed human T cells (Jurkat T cells) wereincubated with soluble humanized VLX4, VLX8, and VLX9 CD47 mAbs (1μg/ml) for 24 hrs at 37° C. As cell death occurs, mitochondrial membranepotential is decreased, the inner leaflet of the cell membrane isinverted, exposing phosphatidylserines (PS), and propidium iodide (PI)is able to incorporate into nuclear DNA. In order to detect thesecellular changes, cells were then stained with fluorescently labeledannexin V and PI or 7-aminoactinomycin D (7-AAD) (BD Biosciences) andthe signal detected using an Accuri C6 flow cytometer (BD Biosciences).The increase in PS exposure is determined by measuring the percentincrease in annexin V signal and the percent of dead cells by measuringthe percent increase in PI or 7-AAD signal. Importantly for therapeuticpurposes, these mAbs induce cell death of tumor cells directly and donot require complement or the intervention of other cells (e.g., NKcells, T cells, or macrophages) to kill. Thus, the mechanism isindependent of both other cells and of Fc effector function. Therefore,therapeutic antibodies developed from these mAbs can be engineered toreduce Fc effector functions such as ADCC and CDC and thereby limit thepotential for side effects common to humanized mAbs with intact Fceffector functions.

As shown in FIG. 12A and FIG. 12B, the soluble VLX4 humanized mAbsinduced cell death of Jurkat T ALL cells as measured by increasedannexin V staining and 7-AAD staining (not shown). The humanized mAbsVLX4hum_01 IgG1, VLX4hum_01 IgG4 PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, andVLX4hum_13 IgG4 PE caused cell death. In contrast, the humanized mAbsVLX4hum_08 IgG4 PE and VLX4hum_11 IgG4 PE did not cause cell death ofJurkat T cells. Induction of cell death and the promotion ofphagocytosis of susceptible cancer cells imparts an additional desirableantibody property and therapeutic benefit in the treatment of cancer.

As shown in FIG. 13A and FIG. 13B, the soluble VLX8 chimeric andhumanized mAbs induced cell death of Jurkat T ALL cells as measured asmeasured by increased annexin V staining and 7-AAD staining (not shown).The chimeric mAbs, VLX8 IgG1 N297Q (xi) and VLX8 IgG4 PE, and thehumanized mAbs, VLX8hum_07 IgG4 PE and VLX8hum_08 IgG4 PE, induced celldeath of Jurkat T ALL cells. In contrast, the humanized mAbs VLX8hum_02IgG4 PE and VLX8hum_04 IgG4 PE did not cause cell death of Jurkat Tcells. Induction of cell death and the promotion of phagocytosis ofsusceptible cancer cells imparts an additional desirable antibodyproperty and therapeutic benefit in the treatment of cancer.

As shown in FIG. 14A, the soluble VLX9 chimeric antibodies induced celldeath of Jurkat cells as measured by increased annexin V staining and7-AAD staining (not shown). In addition as shown in FIG. 14B, thechimeric VLX9 IgG2xi mAb and the humanized mAbs VLX9hum_06 IgG2,VLX9hum_07 IgG2, VLX9hum_08 IgG2, and VLX9hum_09 IgG2 induced cell deathof Jurkat cells (greater than 2-fold increase in annexin V staining). Incontrast, the humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2,VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2 and VLX9hum_010 IgG2did not cause cell death of Jurkat cells. Induction of cell death andthe promotion of phagocytosis of susceptible cancer cells imparts anadditional desirable antibody property and therapeutic benefit in thetreatment of cancer.

Example 7 Hemagglutination of Human Red Blood Cells (hRBCs)

Many CD47 antibodies, including B6H12, BRIC126, MABL1, MABL2, CC2C6,5F9, have been shown to cause hemagglutination (HA) of washed RBCs invitro or in vivo (Petrova P. et al. Cancer Res 2015; 75(15 Suppl):Abstract nr 4271; U.S. Pat. No. 9,045,541; Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract 3019)).Hemagglutination of hRBCs was assessed following incubation of hRBCswith various concentrations of chimeric and humanized VLX4, VLX8, andVLX9 mAbs in vitro essentially as described by Kikuchi et al. BiochemBiophys Res. Commun (2004) 315:912-918. Blood was obtained from healthydonors, diluted (1:50) in PBS/1 mM EDTA/BSA and and washed 3 times withPBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equalvolumes of the antibodies (75 μl of each) and incubated for 3 hrs at 37°C. and overnight at 4° C.

As shown in FIG. 15A and Tables 1 and 2, The VLX4hum_01 IgG1 N297Qcaused hemagglutination of hRBCs, whereas the humanized VLX4hum_01 IgG4PE mAb did not (mAb concentrations 50 μg/ml to 0.3 ng/ml). The lack ofhemagglutination by VLX4hum_01 IgG4 PE imparts an additional desirableantibody property and therapeutic benefit in the treatment of cancer.

As shown in FIG. 15B and Tables 1 and 2, the chimeric antibody VLX8 IgG4PE (xi) and the humanized antibodies VLX8hum_08 IgG4 PE, VLX8hum_09 IgG4PE, and VLX8hum_010 IgG4 PE caused hemagglutination of hRBCs, whereasthe VLX8 humanized Abs VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE,VLX8hum_03 IgG4 PE and VLX8hum_11 IgG4 PE did not (mAb concentrations 50μg/ml to 0.3 ng/ml).

The lack of hemagglutination by humanized antibodies VLX4hum_01 IgG4 PE,VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE andVLX8hum_11 IgG4 PE imparts an additional desirable antibody property anda therapeutic benefit in the treatment of cancer.

As shown in FIG. 16A and FIG. 16B, The chimeric antibody VLX9 IgG2caused hemagglutination of hRBCs, whereas all of the humanized VLX9 mAbsexcept for VLX9hum_07 IgG2, did not (at concentrations from 50 ug/ml to0.3 pg/ml). However, the amount of hemagglutination caused by VLX9hum_07was reduced compared to the VLX9 IgG2 chimeric mAb. Again, the lack ofhemagglutination by the VLX9 humanized mAbs imparts an additionaldesirable antibody property and a therapeutic benefit in the treatmentof cancer.

Example 8 Anti-Tumor Activity In Vivo

The purpose of this experiment was to demonstrate that VLX4, VLX8 andVLX9 humanized antibodies, exemplified by VLX4_07 IgG4PE, VLX8_10 IgG4PEand VLX9hum_08 IgG2, reduce tumor burden in vivo in a mouse xenograftmodel of lymphoma.

Raji human Burkitt's lymphoma cells (ATCC #CCL-86, Manassas, Va.) weremaintained in RPMI-1640 (Lonza; Walkersville, Md.) supplemented with 10%Fetal Bovine Serum (FBS; Omega Scientific; Tarzana, Calif.) within a 5%CO₂ atmosphere. Cultures were expanded in tissue culture flasks.

Female NSG (NOD-Cg-Prkdc_(scid)I12rg^(tm1Wj1)/SzJ) were obtained fromJackson Laboratory (Bar Harbor, Me.) at 5-6 weeks of age. Mice wereacclimated prior to handling and housed in microisolator cages (LabProducts, Seaford, Del.) under specific pathogen-free conditions. Micewere fed Teklad Global Diet® 2920x irradiated laboratory animal diet(Envigo, Formerly Harlan; Indianapolis, Ind.) and provided autoclavedwater ad libitum. All procedures were carried out under InstitutionalAnimal Care and Use guidelines.

Female NSG mice were inoculated subcutaneously in the right flank with0.1 mL of a 30% RPMI/70% Matrigel™ (BD Biosciences; Bedford, Mass.)mixture containing a suspension of 5×10⁶ Raji tumor cells. Five daysfollowing inoculation, digital calipers were used to measure width andlength diameters of the tumor. Tumor volumes were calculated utilizingthe formula: tumor volume (mm³)=(a×b²/2) where ‘b’ is the smallestdiameter and ‘a’ is the largest diameter. Mice with palpable tumorvolumes of 31-74 mm³ were randomized into 8-10/group and VLX9hum_08 orPBS (control) administration was initiated at this time. Mice weretreated with 5 mg/kg of antibody 5×/week for 4 weeks by intraperitonealinjection. Tumor volumes and body weights were recorded twice weekly.

As shown in FIG. 17, treatment with the humanized VLX4hum_07 IgG4 PEsignificantly reduced tumor growth of the Raji tumors (p<0.05, two-wayANOVA), demonstrating anti-tumor efficacy in vivo.

As shown in FIG. 18, treatment with the humanized anti-CD47 mAb,VLX8hum_10 IgG4 PE significantly reduced (p<0.0001, two-way ANOVA) tumorgrowth of the Raji tumors, demonstrating anti-tumor efficacy in vivo.

As shown in FIG. 19, treatment with the humanized anti-CD47 mAb,VLX9hum_08 IgG2 significantly reduced (p<0.05, ANOVA) tumor growth ofthe Raji tumors, demonstrating anti-tumor efficacy in vivo.

Example 9 Effect on Circulating Red Blood Cell Parameters

The purpose of this experiment is to demonstrate that VLX9 humanizedantibodies that do not bind to human RBC in vitro (Table 2), exemplifiedby hum1017_08 IgG2, do not cause a reduction in either hemoglobin (Hg)or circulating RBCs following administration to cynomolgus monkeys.

Female Chinese cynomolgus monkeys (Charles River Laboratories, Houston,Tex.) 2.5-3 kg were used in accordance with the Institutional AnimalCare and Use guidelines. VLX9hum_08 IgG2 or vehicle (PBS) wasadministered as a 1 hour intravenous infusion on day 1 at a dose of 5mg/kg and on day 18 at a dose of 15 mg/kg (3 animals/group).Hematological parameters were measured throughout the study on days −7,−3, 3, 8, 12, 18 (pre-dose), 20, 25, 29, 35 and 41 andcompared/normalized to the means values of control animals. Thepre-treatment RBC and Hg values on day 0 in the VLX9hum_08 IgG2 groupwere lower than the control group. Following treatment with either doseof VLX9hum_08 IgG2, there were minimal changes (<10%) in Hg (FIG. 20A)or RBC counts (FIG. 20B) compared to the control demonstrating thatantibodies that do not bind to human RBCs in vitro do not cause areduction in RBC hematological parameters when administered tocynomolgus monkeys.

Example 10 Immunohistochemical Staining of CD47

Localization of CD47 expression was determined in formalin-fixed,paraffin embedded (FFPE) blocks from patients with a number of types ofcancer (obtained from commercial sources) using mouse/rabbit chimericanti-CD47 mAbs. 3-4 micron sections were cut from FFPE blocks,deparaffinized and treated with antigen retrieval solution. Sectionswere then incubated with 4 μg/ml of the primary anti-CD47 mouse/rabbitchimeric mAbs for 1 hr and with an anti-rabbit HRP labeled secondaryantibody for 20 minutes. The anti-CD47 antibody bound to human CD47 wasvisualized using the peroxidase substrate,3,3′,5,5′-tetramethylbenzidene. Sections were counterstained withhematoxylin and evaluated using standard light microscopy. As shown inFIG. 21, high CD47 expression was detected in human breast cancertissue, as shown by dark areas denoted by arrows, using CD47mouse/rabbit chimeric mAbs, exemplified by the VLX4 mouse/rabbitchimeric mAb. This demonstrates that these mAbs can be used forimmunohistochemical localization of human CD47 in tumor tissue sectionsobtained from FFPE blocks in diagnostic assays.

Example 11 Antibodies to CD47 Regulate Nitric Oxide Signaling

TSP1 binding to CD47 activates the heterotrimeric G protein Gi, whichleads to suppression of intracellular cyclic AMP (cAMP) levels. Inaddition, the TSP1/CD47 pathway opposes the beneficial effects of thenitric oxide (NO) pathway in all vascular cells. The NO pathway consistsof any of three nitric oxide synthase enzymes (NOS I, NOS II and NOSIII) that generate bioactive gas NO using arginine as a substrate. NOcan act within the cell in which it is produced or in neighboring cells,to activate the enzyme soluble guanylyl cyclase that produces themessenger molecule cyclic GMP (cGMP). The proper functioning of theNO/cGMP pathway is essential for protecting the cardiovascular systemagainst stresses including, but not limited to, those resulting fromwounding, inflammation, hypertension, metabolic syndrome, ischemia, andischemia-reperfusion injury (IRI). In the context of these cellularstresses, the inhibition of the NO/cGMP pathway by the TSP1/CD47 systemexacerbates the effects of stress. This is a particular problem in thecardiovascular system where both cGMP and cAMP play important protectiveroles. There are many cases in which ischemia and reperfusion injurycause or contribute to disease, trauma, and poor outcomes of surgicalprocedures.

The purpose of these experiment will be to demonstrate that humanizedanti-CD47 mAbs of the present disclosure exhibit the ability to reverseTSP1-mediated inhibition of NO-stimulated cGMP synthesis as, forexample, described previously using mouse monoclonal antibodies to CD47as disclosed by Isenberg et al. (2006) J. Biol. Chem. 281:26069-80, oralternatively other downstream markers of or effects resulting from NOsignaling, for example smooth muscle cell relaxation or plateletaggregation as described previously by Miller et al. (2010) Br J.Pharmacol. 159: 1542-1547.

The method employed that will be to measure cGMP as described by themanufacturer (CatchPoint Cyclic-GMP Fluorescent Assay Kit, MolecularDevices, Sunnyvale, Calif.). Jurkat JE6.1 cells (ATCC, Manassas, Va.;Catalog # TIB-152) or other cells types that retain the NO/cGMPsignaling pathway when grown in culture and exhibit a robust andreproducible inhibitory response to TSP1 ligation of CD47 will be used.Cells will be grown in Iscove's modified Dulbeccco's medium containing5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue #S01520), 100 units/mL penicillin, 100 μg mL streptomycin (Sigma;Catalogue # P4222) at densities less than 1×106 cells/mL. For the cGMPassay, cells will be plated in 96 well tissue culture plates at adensity of 1×10⁵ cells/ml in Iscoves modified Dulbecco's mediumcontaining 5% (v/v) heat inactivated fetal bovine serum (BioWest;Catalog # S01520), 100 units/mL penicillin, 100 μg/mL streptomycin(Sigma; #P4222) for 24 hours and then transferred to serum free mediumovernight.

The humanized antibodies as disclosed herein, purified from transienttransfections in CHO cells as described above in Example 3, as well asthe control chimeric antibody, will then be added at a finalconcentration of 20 ng/ml, followed 15 minutes later by 0 or 1 μg/mlhuman TSP1 (Athens Research and Technology, Athens, Ga., Catalogue#16-20-201319). After an additional 15 minutes, the NO donor,diethylamine (DEA) NONOate (Cayman Chemical, Ann Arbor, Mich., Catalog#82100), will be added to half the wells at a final concentration of 1Five minutes later, the cells will be lysed with buffer supplied in thecGMP kit, and aliquots of each well assayed for cGMP content.

It is anticipated that some of the chimeric or humanized antibodies willreverse TSP1 inhibition of cGMP. Reversal will be complete (>80%) orintermediate (20%-80%). This reversal of TSP1 inhibition of cGMP willdemonstrate that they have the ability to increase NO signaling andsuggest utility in protecting the cardiovascular system against stressesincluding, but not limited to, those resulting from wounding,inflammation, hypertension, metabolic syndrome, ischemia, andischemia-reperfusion injury (IRI). Additional assay systems, for examplesmooth muscle cell contraction, will also be expected to show that someof the chimeric or humanized antibody clones reverse the inhibitoryactions of TSP on downstream effects resulting from the activation of NOsignaling.

What is claimed:
 1. A monoclonal antibody, or antigen-binding fragmentthereof, that specifically binds CD47 and comprises: a variable heavychain CDR1 amino acid sequence (HCDR1) amino acid sequence set forth inSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; a variable heavy chain CDR2amino acid sequence (HCDR2) amino acid sequence set forth in SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6; a variable heavy chain CDR3 aminoacid sequence (HCDR3) amino acid sequence set forth in SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9 or SEQ ID NO:10; a variable light chain CDR1 aminoacid sequence (LCDR1) amino acid sequence set forth in SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, or SEQ ID NO:14; a variable light chain CDR2amino acid sequence (LCDR2) amino acid sequence set forth in SEQ IDNO:15, SEQ ID NO:16, or SEQ ID NO:17; and a variable light chain CDR3amino acid sequence (LCDR3) amino acid sequence set forth in SEQ IDNO:18, SEQ ID NO:19, or SEQ ID NO:20.
 2. The monoclonal antibody, orantigen-binding fragment thereof, of claim 1, wherein the monoclonalantibody, or antigen-binding fragment thereof is chimeric or humanized.3. The monoclonal antibody, or antigen-binding fragment thereof, ofclaim 1, wherein the monoclonal antibody, or antigen-binding fragmentthereof specifically binds human CD47.
 4. The monoclonal antibody, orantigen-binding fragment thereof, of claim 3, wherein the monoclonalantibody, or antigen-binding fragment thereof specifically binds atleast one of cynomolgus monkey, rat, mouse, or pig CD47.
 5. Themonoclonal antibody, or antigen-binding fragment thereof, of claim 1,further comprising a heavy chain variable domain (VH) and a light chainvariable domain (VL), selected from the group consisting of: (i) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:21and a light chain variable domain comprising the amino acid sequence SEQID NO:41; (ii) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:23 and a light chain variable domain comprisingthe amino acid sequence SEQ ID NO:43; (iii) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:24 and a lightchain variable domain comprising the amino acid sequence SEQ ID NO:43;(iv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:26 and a light chain variable domain comprising the amino acidsequence SEQ ID NO:43; (v) a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO:26 and a light chain variable domaincomprising the amino acid sequence SEQ ID NO:44; (vi) a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO:27 and alight chain variable domain comprising the amino acid sequence SEQ IDNO:43; (vii) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:27 and a light chain variable domain comprisingthe amino acid sequence SEQ ID NO:44; (viii) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:28 and a lightchain variable domain comprising the amino acid sequence SEQ ID NO:46;(ix) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:29 and a light chain variable domain comprising the amino acidsequence SEQ ID NO:47; (x) a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO:29 and a light chain variable domaincomprising the amino acid sequence SEQ ID NO:48; (xi) a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO:30 and alight chain variable domain comprising the amino acid sequence SEQ IDNO:47; (xii) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:30 and a light chain variable domain comprisingthe amino acid sequence SEQ ID NO:48; (xiii) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:31 and a lightchain variable domain comprising the amino acid sequence SEQ ID NO:47;(xiv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:31 and a light chain variable domain comprising the aminoacid sequence SEQ ID NO:48; (xv) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:32 and a light chainvariable domain comprising the amino acid sequence SEQ ID NO:47; (xvi) aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO:32 and a light chain variable domain comprising the amino acidsequence SEQ ID NO:48; (xvii) a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO:33 and a light chain variabledomain comprising the amino acid sequence SEQ ID NO:47; (xviii) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:33and a light chain variable domain comprising the amino acid sequence SEQID NO:48; (xix) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:34 and a light chain variable domain comprisingthe amino acid sequence SEQ ID NO:49; (xx) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:35 and a light chainvariable domain comprising the amino acid sequence SEQ ID NO:50; (xxi) aheavy chain variable domain comprising the amino acid sequence of SEQ IDNO:36 and a light chain variable domain comprising the amino acidsequence SEQ ID NO:51; (xxii) a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO:36 and a light chain variabledomain comprising the amino acid sequence SEQ ID NO:52; (xxiii) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:37and a light chain variable domain comprising the amino acid sequence SEQID NO:51; (xxiv) a heavy chain variable domain comprising the amino acidsequence SEQ ID NO:37 and a light chain variable domain comprising theamino acid sequence SEQ ID NO:52; (xxv) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:38 and a light chainvariable domain comprising the amino acid sequence SEQ ID NO:51; (xxvi)a heavy chain variable domain comprising the amino acid sequence of SEQID NO:38 and a light chain variable domain comprising the amino acidsequence SEQ ID NO:52; (xxvii) a heavy chain variable domain comprisingthe amino acid sequence of SEQ ID NO:39 and a light chain variabledomain comprising the amino acid sequence SEQ ID NO:51; (xxviii) a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:39and a light chain variable domain comprising the amino acid sequence SEQID NO:52; (xxix) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:40 and a light chain variable domain comprisingthe amino acid sequence SEQ ID NO:51; and (xxx) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:40 and a lightchain variable domain comprising the amino acid sequence SEQ ID NO:52.6. A pharmaceutical composition, comprising the monoclonal antibody, orantigen-binding fragment thereof, of claim 1, and a pharmaceutically orphysiologically acceptable carrier, diluent, or excipient.